Name: ovarian tissue culture to visualize phenomena in mouse ovary
Uploaded: 2018-06-19T14:30:00.000+00:00
Duration: 4 min 30 s
Description: Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Ringraziamo il Dr. Naojiro Minami (Kyoto University) per fornire i topi Oog1pro3.9 . Questa ricerca è stata sostenuta da JSP (KAKENHI # JP15H06275) e la Fondazione di Nitto.

Topi sono stati alloggiati in una stanza in ambiente controllata a 23 ± 1 ° C con un ciclo di 12 h luce/12 h scuro. Protocolli di cura degli animali e gli esperimenti sono stati condotti in conformità con le linee guida per la sperimentazione animale all'Università di Aichi Medical e sono stati approvati dal comitato di uso e Animal Care incombente.
1. preparazione del terreno di coltura e piatti
<ol><li>Per preparare il terreno di coltura di base, aggiungere siero bovino fetale (FBS, 5% v/v), FSH (100 mIU/mL), LH (10 mU/mL) e la penicillina-streptomicina (penicillina, 100 U/mL; streptomicina, 100 mg/mL) al minimo essenziale medio (MEM-alfa) e mix in provette da 50 mL. Nota: Volumi totali di terreni di coltura richiesto variano fra gli esperimenti, ma 1 mL di terreno di coltura è generalmente sufficiente per piastre di coltura di 3,5 cm con fondo di vetro.</li>
	<li>Versare le aliquote di 1 mL di terreno di coltura mista in piastre di coltura di 3,5 cm con fondo di vetro e set posto 30 mm cella cultura inserti in piatti. Preriscaldare la media e piatti in un'incubatrice (5% CO2 e 37 ° C).</li>
</ol>2. preparazione del tessuto ovarico
<ol><li>Pre-riscaldare tampone salino (PBS) (−) e MEM-alfa contenente 5% FBS, 100 U/mL di penicillina e streptomicina 100 mg/mL in un incubatore (5% CO2 e 37 ° C). Nota: 3 mL circa aliquote di PBS (−) per ovaia sono sufficienti per l'utilizzo durante le dissezioni dell'ovaia, e le aliquote di 1ml di MEM-alfa sono sufficienti per l'archiviazione temporanea delle ovaie singole in piatti di 3,5 cm.</li>
	<li>
Asportare le ovaie da topi ICR (denominata dopo l'Istituto di ricerca sul cancro) femmina 4 - settimana-vecchi e per tagliare i tessuti che circondano l'ovario con delle forbici, pinzette sotto un microscopio stereoscopico. Riserviamo le ovaie rimosse nel terreno di coltura (Vedi punto 2.1) fino all'utilizzo.
	<ol>
<li>Posto singole ovaie su carta da filtro inumidite con pre-riscaldati PBS (−) (Vedi punto 2.1) e tagliare in 4 pezzi usando una lama del microtomo sotto un microscopio stereoscopico. Nota: Ovaie di topi ICR 4 - settimana-vecchi sono circa 2 mm di diametro. Il numero di pezzi dipende dalle dimensioni dell'ovaio; Tuttavia, circa 500 µm spessore pezzi consentono per le osservazioni del follicolo corretta (in pezzi di spessore superiore a 500 mm, trasparenza del tessuto è diminuito; in pezzi più sottile di 500mm, molti follicoli antrali sono rotti e caduti).</li>
		<li>Dopo la dissezione, collocare gli esemplari di affettato ovaio in terreno di coltura pre-riscaldato a piatti di 3,5 cm (Vedi punto 2.1).</li>
	</ol>
</li>
</ol>3. ovarico coltura tissutale
<ol><li>Goccia circa 0,5 µ l di terreno di coltura per affettato tessuto ovarico sull'inserto di cultura cellulare dove il tessuto ovarico sarà impostato utilizzando una micropipetta.</li>
	<li>Posizionare ogni provino a fette in una goccia di terreno di coltura sugli inserti di cultura cellulare usando la pinzetta.</li>
	<li>Cultura dei tessuti dell'ovaia in 5% CO2 a 37 ° C.</li>
	<li>Sostituire il terreno di coltura con medium fresco pre-riscaldato ogni 2 giorni. Nota: La pianificazione per i cambiamenti medi per la cultura delle sezioni dell'ovaia da 4 - settimana-vecchi topi ICR è presentata nella Figura 1.<ol>
<li>Per riprodurre l'impulso del LH, trattare coltivate le ovaie di topi ICR 4 - settimana-vecchie con il mezzo contenente 100 mU/mL FSH e LH per 12 h ogni 4 giorni (Figura 1).</li>
	</ol>
</li>
</ol>4. microscopio immagini delle ovaie coltivate
<ol><li>Avviare le ovaie colte di imaging dopo 1 giorni quando i tessuti hanno aderito sugli inserti di cultura cellulare ed eseguire analisi microscopio confocale o invertito a intervalli di 24 ore per consentire la crescita del follicolo sufficiente tra i punti di tempo. Nota: La microscopia confocale è superiore alla microscopia invertita per osservare follicoli nelle ovaie tutto coltivate.</li>
</ol>5. Time-lapse Imaging delle ovaie coltivate
<ol><li>
Ottimizzare le condizioni di imaging time-lapse, tra cui laser intensità e tempi di esposizione, per il sistema di imaging (tabella 1).
	<ol>
<li>Selezionare gli esemplari accoppiati ovaia da topi singolo per uso come gruppi di controllo e di trattamento. Variare l'intensità del laser e tempo di esposizione per ottenere le migliori immagini (tabella 1).</li>
		<li>Confrontare la crescita del follicolo in ciascuna condizione di cultura misurando aree follicolo in immagini di campioni di controllo a intervalli di 24 h e in time-lapse campioni imaging (Vedi punto 6). Contemporaneamente, contare il numero di ovociti ovulati in ogni set di ovaio e scegliere le condizioni ottimali di imaging time-lapse.</li>
	</ol>
</li>
	<li>Acquisizione di immagini ad intervalli di 30 min con il sistema di imaging time-lapse sotto determinate condizioni (tabella 1).</li>
</ol>6. analisi di crescita del follicolo
<ol><li>
Per analizzare lo sviluppo del follicolo, misurare le aree di follicolo in immagini catturate utilizzando software ImageJ (http://resbweb.nih.gov/ij/).
	<ol>
<li>Inizialmente, è possibile impostare la scala dell'immagine facendo clic su "Imposta scala" sotto "Analizza" nella barra degli strumenti. Immettere le lunghezze laterali dell'immagine catturata e i corrispondenti numeri di pixel nei campi vuoti di "Distanza di noto" e "Distanza in pixel", rispettivamente.</li>
		<li>Fare clic su "Mano libera" nella barra degli strumenti e delineare i follicoli nelle immagini catturate campo luminoso.</li>
		<li>Nella barra degli strumenti, fare clic su "Misura" sotto "Analizza". Nota: Se altri dati di misura sono desiderati, fare clic su "Set misura" sotto "Analizza" nella barra degli strumenti e seleziona le caselle appropriate nell'elenco.</li>
	</ol>
</li>
</ol>7. analisi dello sviluppo del follicolo utilizzando topi transgenici
<ol><li>Raccogliere le ovaie da giorni postnatali 0 e 4 (P0 e P4) femmina topi transgenici contenenti i transgeni Oog1pro3.9 e R26-H2B-mCherre cultura su inserti come descritto passaggi 1.1 – 3.2, tranne non fetta P0 e ovaie P4.</li>
	<li>Sostituire il terreno di coltura con medium fresco, pre-riscaldato in piatti di 3,5 cm in un'incubatrice che contiene 5% CO2 a 37 ° C ogni 2 giorni. Nota: La concentrazione di LH nel terreno di coltura delle ovaie P0 e P4 può rimanere costante perché picchi di LH non si verificano nei topi P0 e P4.</li>
	<li>Impostare il microscopio per visualizzare solo follicoli primari di AcGFP1-positivi in coltura P4 ovaie (tabella 1). Nota: Solo primordiale e primari follicoli sono presenti nelle ovaie femminili del mouse P4.</li>
	<li>Catturare immagini di colta P0 Oog1pro3.9/ ovaie di topi transgeniciR26-H2B-mCherry ad intervalli di 30 min utilizzando le impostazioni utilizzate per P4 ovaie (Vedi punto 5.2).</li>
	<li>Tracciare e analizzare lo sviluppo del follicolo utilizzando segnali AcGFP1 e H2B-mCHerry.</li>
</ol>

<ol>
	<li>Myers, M., Britt, K. L., Wreford, N. G., Ebling, F. J., Kerr, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+follicular+numbers+within+the+mouse+ovary.">Methods for quantifying follicular numbers within the mouse ovary.</a> Reproduction. 127 (5), 569-580 (2004).</li><li>Adams, G. P., Jaiswal, R., Singh, J., Malhi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+ovarian+follicular+dynamics+in+cattle.">Progress in understanding ovarian follicular dynamics in cattle.</a> Theriogenology. 69 (1), 72-80 (2008).</li><li>Palma, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+biotechnology+of+follicle+development.">Biology and biotechnology of follicle development.</a> Sci World J. , (2012).</li><li>Knight, P. G., Glister, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+superfamily+members+and+ovarian+follicle+development.">TGF-beta superfamily members and ovarian follicle development.</a> Reproduction. 132 (2), 191-206 (2006).</li><li>Picton, H. M., Harris, S. E., Muruvi, W., Chambers, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+growth+and+maturation+of+follicles.">The in vitro growth and maturation of follicles.</a> Reproduction. 136 (6), 703-715 (2008).</li><li>Spears, N., de Bruin, J. P., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+follicular+dominance+in+co-cultured+mouse+ovarian+follicles.">The establishment of follicular dominance in co-cultured mouse ovarian follicles.</a> J Reprod Fertil. 106 (1), 1-6 (1996).</li><li>Baker, S. J., Srsen, V., Lapping, R., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effect+of+follicle-follicle+interactions+and+declining+follicle-stimulating+hormone+on+murine+follicle+health+in+vitro.">Combined effect of follicle-follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro.</a> Biol Reprod. 65 (4), 1304-1310 (2001).</li><li>Komatsu, 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=Analysis+of+the+Effect+of+Leukemia+Inhibitory+Factor+on+Follicular+Growth+in+Cultured+Murine+Ovarian+Tissue.">Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue.</a> Biol Reprod. 93 (1), 18 (2015).</li><li>Komatsu, K., Masubuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration-dependent+effect+of+progesterone+on+follicle+growth+in+the+mouse+ovary.">The concentration-dependent effect of progesterone on follicle growth in the mouse ovary.</a> J Reprod Dev. 63 (3), 271-277 (2017).</li><li>Murase, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+dynamics:+visualization+and+analysis+of+follicle+growth+and+maturation+using+murine+ovarian+tissue+culture.">Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture.</a> J Assist Reprod Genet. , 1-5 (2017).</li><li>Ishida, 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=The+promoter+of+the+oocyte-specific+gene,+Oog1,+functions+in+both+male+and+female+meiotic+germ+cells+in+transgenic+mice.">The promoter of the oocyte-specific gene, Oog1, functions in both male and female meiotic germ cells in transgenic mice.</a> PLoS One. 8 (7), 68686 (2013).</li><li>Minami, N., 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=Oogenesin+is+a+novel+mouse+protein+expressed+in+oocytes+and+early+cleavage-stage+embryos.">Oogenesin is a novel mouse protein expressed in oocytes and early cleavage-stage embryos.</a> Biol Reprod. 69 (5), 1736-1742 (2003).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+factor-9+stimulates+progression+of+early+primary+but+not+primordial+rat+ovarian+follicle+development.">Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.</a> Biol Reprod. 67 (3), 1018-1024 (2002).</li><li>Nilsson, E. E., Kezele, P., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia+inhibitory+factor+(LIF)+promotes+the+primordial+to+primary+follicle+transition+in+rat+ovaries.">Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries.</a> Mol Cell Endocrinol. 188 (1-2), 65-73 (2002).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-4+acts+as+an+ovarian+follicle+survival+factor+and+promotes+primordial+follicle+development.">Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.</a> Biol Reprod. 69 (4), 1265-1272 (2003).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kit+ligand+and+basic+fibroblast+growth+factor+interactions+in+the+induction+of+ovarian+primordial+to+primary+follicle+transition.">Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition.</a> Mol Cell Endocrinol. 214 (1-2), 19-25 (2004).</li><li>Abe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+conditional+reporter+mouse+lines+at+ROSA26+locus+for+live+cell+imaging.">Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging.</a> Genesis. 49 (7), 579-590 (2011).</li><li>Lan, Z. J., Xu, X., Cooney, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+oocyte-specific+expression+of+Cre+recombinase+activity+in+GDF-9-iCre,+Zp3cre,+and+Msx2Cre+transgenic+mice.">Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice.</a> Biol Reprod. 71 (5), 1469-1474 (2004).</li><li>Payer, B., 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=Generation+of+stella-GFP+transgenic+mice:+a+novel+tool+to+study+germ+cell+development.">Generation of stella-GFP transgenic mice: a novel tool to study germ cell development.</a> Genesis. 44 (2), 75-83 (2006).</li><li>Ohinata, Y., Sano, M., Shigeta, M., Yamanaka, K., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive,+non-invasive+visualization+of+primordial+germ+cell+development+in+mice+by+the+Prdm1-mVenus+and+Dppa3-ECFP+double+transgenic+reporter.">A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter.</a> Reproduction. 136 (4), 503-514 (2008).</li></ol>

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In questo studio, abbiamo sviluppato due nuovi metodi per studiare lo sviluppo del follicolo nelle ovaie del mouse. Il primo metodo comporta la cultura dei tessuti di topi adulti ovarico affettato seguita da analisi dello sviluppo del follicolo, e il secondo prevede l'utilizzo di time-lapse imaging per visualizzare lo sviluppo iniziale del follicolo durante la fase di gonadotropina-indipendente. In precedenza, abbiamo usato il presente metodo di coltura del tessuto ovario per valutare l'effetto di fattore inibitorio di l...

Mouse femminile ovaie contengono parecchie migliaia di follicoli1, e l'ovulazione periodica matura circa dieci ovociti ad ogni ciclo di estro. Follicoli sono classificati in diversi stadi di sviluppo: primordial, primaria, secondaria, antral e follicoli di Graaf, a seconda della forma dello strato cellulare granulosa che circondano ogni ovocita. Maggior parte dei follicoli primordiali sono dormienti, e alcuni di essi sono attivati e crescere in follicoli primari a ogni ciclo di estro2. Dopo la fase follicolare secondaria, sviluppo del follicolo è regolato principalmente da gonadotropine, follicolare di stimolazione dell'ormone (FSH) e luteinizzante (LH). Tuttavia, lo sviluppo del follicolo primordiale e primario è indipendente della gonadotropina e i meccanismi regolatori che governano queste fasi rimangono scarsamente inderstood3,4,5. Oltre ai fattori di crescita e ormoni, il follicolo primordiale e primario è regolato dalle interazioni tra follicoli6,7. Pertanto, abbiamo eseguito le analisi delle dinamiche del follicolo in tessuti di topo dell'ovaia e studiato i meccanismi normativi associati utilizzando colture del tessuto ovarico8,9,10.
Qui, presentiamo due metodi di modello di coltura del tessuto ovarico. Il primo viene utilizzato per analizzare lo sviluppo del follicolo tramite la misura delle aree follicolare, e il secondo è usato per studiare il meccanismo regolatore durante lo sviluppo iniziale di follicolo da primordiale alla fase del follicolo secondario con topi transgenici. Per l'analisi di sviluppo del follicolo, abbiamo usato soprattutto le ovaie di topi femminili 4 - settimana-vecchi perché consentono per una visualizzazione semplice dei follicoli. Per indurre l'ovulazione periodica e modello in vivo lo sviluppo del follicolo, abbiamo riprodotto il picco di LH e osservato l'ovulazione, l'atresia del follicolo e secrezione dell'estradiolo in condizioni di coltura del tessuto. Immagini delle ovaie coltivate furono catturati, e i processi di sviluppo del follicolo sono stati analizzati dai cambiamenti di tracciatura in zona follicolare. Tuttavia, nelle analisi di microscopia del campo luminoso, la distinzione tra follicoli primari primordiale e all'inizio era poco chiara. Così, abbiamo sviluppato un metodo per rilevare piccoli follicoli e distinguere tra la primordiale, primaria, e follicolo secondario in coltivate ovarici tessuti utilizzando Oogenesin1 (Oog1) pro3. 9 e ovaie di topi transgenici R26-H2B-mCherry ai giorni 0 e 4 dopo nascita11. Oog1 espressione è rilevabile negli ovociti dopo l'entrata in meiosi e aumenta gradualmente con lo sviluppo del follicolo, permettendo l'osservazione della transizione da primordial follicoli primari utilizzando immagini time-lapse del tessuto ovario coltivate11 ,12. Anche se i metodi morfologici sono stati utilizzati per studiare i fattori che attivano i follicoli primordiali dormente13,14,15,16, lo sviluppo fisiologico del follicolo nelle ovaie è difficile da osservare, e gli effetti di vari fattori rimangono atipici. Gli attuali metodi di coltura sono stati progettati per affrontare questa scarsità in tempo reale analisi dei fattori di destinazione.
Nello studio presente, abbiamo rintracciato lo sviluppo follicolare, utilizzando un metodo di imaging time-lapse e caratterizzato il processo di sviluppo del follicolo. I nostri metodi innovativi offrono uno strumento senza precedenti per studiare la fisiologia delle ovaie.

<table><tbody><tr><td>Follicle stimulating hormone from human pituitary</td><td>SIGMA</td><td>F4021</td><td></td></tr><tr><td>Lutenizing hormone from equine pituitary</td><td>SIGMA</td><td>L9773</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>Wako Pure Chemical Industries</td><td>168-23191</td><td></td></tr><tr><td>MEM a, GlutaMax, no nucleotides</td><td>Thermo Fisher</td><td>32561037</td><td></td></tr><tr><td>Glass bottom dish</td><td>MatTek</td><td>P35G-0-10-C</td><td>35mm dish, No. 0 coverslip, 10mm glass diameter</td></tr><tr><td>Millicell cell culture insert</td><td>Merck Millipore</td><td>PICM0RG50</td><td>Diameter: 315 mm, pore size: 0.4 mm, material: hydrophilic PTTE</td></tr><tr><td>3.5cm cell culture dishes</td><td>greiner bio-one</td><td>627160</td><td></td></tr><tr><td>50ml / centrifuge tube with triple seal cap</td><td>IWAKI</td><td>2345-050</td><td></td></tr><tr><td>Low-profile disposable blades 819</td><td>Leica</td><td>14035838925</td><td></td></tr><tr><td>LSM 710</td><td>Carl Zeiss</td><td></td><td>Confocal microscope</td></tr><tr><td>CellVoyager, CV1000</td><td>Yokogawa Electric Corporation</td><td></td><td>Time-lapse imaging</td></tr><tr><td>BZ-X700</td><td>KEYENCE</td><td></td><td>Time-lapse imaging</td></tr></tbody></table>

ovarian tissue culture to visualize phenomena in mouse ovary

Le femmine dei mammiferi ovulano periodicamente un numero quasi costante degli ovociti durante ogni ciclo di estro. Per sostenere tale regolarità e periodicità, la regolazione avviene a livello ipotalamico-pituitario-gonadico e sullo sviluppo di follicoli nell'ovaio. Nonostante studi attiva, meccanismi per lo sviluppo del follicolo non sono chiari a causa dei diversi passaggi coinvolti dall'attivazione dormienti follicolo primordiale all'ovulazione e a causa della complessità del regolamento che è diverso in ogni fase follicolare. Per studiare i meccanismi di sviluppo del follicolo e le dinamiche dei follicoli durante tutto il ciclo di estro, abbiamo sviluppato un modello di coltura del tessuto ovarico del mouse che può essere usato per osservare lo sviluppo del follicolo tramite un microscopio. Sviluppo sistematico del follicolo, l'ovulazione periodica ed atresia del follicolo può essere riprodotto nel modello dell'ovaia coltivate, e le condizioni della coltura possono essere modulate sperimentalmente. Qui, noi dimostrare l'utilità di questo metodo nello studio dei meccanismi di regolazione dello sviluppo del follicolo e altri fenomeni ovarici.

La figura 1 Mostra il protocollo per i cambiamenti nei mezzi di comunicazione durante la coltura del tessuto ovarico. Seguendo questo programma, le ovaie di topi ICR 4-settimana-vecchio erano coltivate e ripreso a intervalli di 24-h usando la microscopia confocale (Figura 2). Durante la coltura dei tessuti dell'ovaia per 3 settimane, più follicoli antrali e secondari sono stati degenerati da atresia del follicolo e alcuni erano ...

Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Coltura del tessuto ovarico per visualizzare fenomeni nell'ovaia di Mouse

Colture del tessuto ovarici possono essere utilizzati come modelli di atresia di sviluppo, l'ovulazione e follicolo pilifero e indicare i meccanismi regolatori dei processi dinamici ovarici.

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

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13.7K Views.  Aichi Medical University. Colture del tessuto ovarici possono essere utilizzati come modelli di atresia di sviluppo, l'ovulazione e follicolo pilifero e indicare i meccanismi regolatori dei processi dinamici ovarici.

Video: Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

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.

Cultura e co-coltura di mouse ovaie e follicoli ovarici

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

Ricerca

Biologia dello sviluppo

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Un modello ortotopico di carcinoma ovarico sieroso in topi immunocompetenti per In vivo Tumore Imaging e monitoraggio del tumore Risposte immunitarie

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunologia e infezioni

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Bioingegneria

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Tutta Monte immunofluorescenza e quantificazione del follicolo delle ovaie coltivati del topo

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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.

Enumerazione accurata del follicolo nelle ovaie di topo adulto

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

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Biologia

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

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

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

Immunofluorescenza dell'ovaio intero, clearing e microscopia multifotonica per l'analisi quantitativa 3D della riserva ovarica in via di sviluppo nel topo

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

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Ricostruzione tridimensionale dell'architettura vascolare dell'ovaia passiva sdoganate per la chiarezza del Mouse

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

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.

Coltura tissutale della corteccia ovarica bovina

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

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

To carry out orthotopic inoculation, after anesthetizing an eight-week-old female nude mouse according to the text protocol, perform a toe pinch to verify depth of the anesthesia. Place the anesthetized mouse on an operating stage, belly side down. Then, disinfect the surgical area with several washes using a chlorhexidine-based scrub and alcohol.
Next, make an approximately 2-centimeter vertical incision near the spine, between the right costal arch and the femur. Place a sterile surgical drape around the incision site. Then, fix the incised skin surfaces with clamps to maintain the opening.
Now, make a second 1-centimeter incision at the parietal peritoneum under the first incision to open the abdominal cavity. Then, clamp the incised abdominal surfaces. With a clamp, clip the fat tissue that surrounds the ipsilateral ovary and fallopian tube to remove the ovary from the abdominal cavity.
Then, carefully place the ovary and fallopian tube on gauze before placing the mouse, belly side down, under the dissecting microscope. Transfer the cell suspension to an insulin syringe. Then, immediately and carefully inject 1 to 2 microliters of the prepared cell suspension into the ovary.
For several seconds, keep the needle in the ovary to ensure that the hydrogel sets. After gelation, the injected tumor cells will remain in the ovary. Carefully, return the ovary into the body. With sterile medical silk, suture the second incision before using wound clips to seal the skin.

Inoculazione ortotopica di cellule di cancro ovarico nell'ovaio murino: una procedura per stabilire un modello murino di cancro ovarico per studiare la disseminazione peritoneale delle cellule