We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

Alle folgten die Verfahren in dieser Studie mit der Association for Research gewährt in Vision and Ophthalmology Erklärung für die Verwendung von Tieren in Ophthalmic und Vision Research und wurden von der Institutional Animal Care und Use Committee der National Taiwan University Hospital genehmigt. 1. Isolierung, Primärkultur Vorbereitung, und Immunfärbung von Bovine MEL <ol><li> Erwerben Sie frische Rinderaugen von einem lokalen Schlachthof. </li><li> Desinfizieren der Augen in einer 10% w / v Povidon-Iod-Lösung für 3 min. Wasche sie mit phosphatgepufferter Kochsalzlösung (PBS) -Lösung. </li><li> Ernten Sie die Hornhaut-Taste mit einem Skalpell und Schere unter sterilen Bedingungen. Ziehen Sie die Descemet-Membran mit einer Pinzette unter einem Binokular (beachten Sie, dass in dieser Studie wurden die Rinderaugen von einem örtlichen Schlachthof entkernte, daher ist die erste Studie Verfahren war die Desinfektion der preenucleated Augen im Labor). </li><li> Inkubieren der Membran Descemet in 1 ml tryPSIN bei 37 ° C für 30 min. Sammeln Sie die Rinder MEL durch Zentrifugation bei 112 × g für 5 min. Resuspendieren der Zellen in 1 ml supplementiert hormonellen epithelial Medium (SHEM) gleiche Volumina von HEPES-gepufferter Dulbecco-modifiziertem Eagle-Medium und Ham-F12-Medium, ergänzt mit 5% fötalem Rinderserum, 0,5% Dimethylsulfoxid, 2 ng / ml des humanen epidermalen Wachstumsfaktor, 5 mg / ml Insulin, 5 mg / ml Transferrin, 5 ng / ml Selen, 1 nmol / l des Choleratoxins, 50 mg / ml Gentamicin und 1,25 mg / ml Amphotericin B. </li><li> Seed die Zellen (etwa 1 x 10 5 Zellen pro Auge) in eine 6 cm Schale. Kultur sie in der SHEM. Inkubiere die Schale bei 37 ° C in einer Atmosphäre von 5% CO 2 in Luft. Ändern Sie das Kulturmedium alle 3 Tage. </li><li> Wenn die Zellen Konfluenz erreichen, waschen Sie sie in PBS und Inkubation sie in 1 ml Trypsin bei 37 ° C für 5 min. Sammeln Sie sie durch Zentrifugation bei 112 × g für 5 min. Resuspendieren des Zellpellets in 1 ml des SHEM. <ol&gt; <li> Zählen Sie die Zellen in einem Hämozytometer. Seed die Zellen auf Deckgläser mit einer Dichte von 1 x 10 4 pro Well in einer 24-Well - Platte und Kultur die Zellen in der SHEM. die ENMT Unterdrückungseffekt von Marimastat zur Untersuchung, inkubiere die Zellen in SHEM mit 10 uM Marimastat in das Kulturmedium gegeben, und das Kulturmedium alle 3 Tage. </li></ol></li><li> Fixieren Sie die Zellen in einem angegebenen Zeitpunkt mit 250 ul 4% Paraformaldehyd, pH 7,4, für 30 min bei Raumtemperatur. Permeabilisieren mit 250 ul 0,5% Triton X-100 5 min. Block mit 10% Rinderserumalbumin für 30 min. </li><li> Inkubieren Sie die Zellen mit primären Antikörpern gegen aktive Beta-Catenin (ABC), Schnecke und Schnecke über Nacht bei 4 ° C. Die Verdünnung der primären Antikörpern verwendet wird, ist 1: 200. Die Antikörper werden in Antikörperverdünnungslösung, bestehend aus 10 mM PBS, 1% w / v Rinderserumalbumin und 0,09% w / v Natriumazid verdünnt. </li><li> Waschen Sie die Zellen zweimal mit PBS für 15 min und Inkubation derm mit dem Alexa Fluor-konjugiertem sekundärem Antikörper (1: 100 in Antikörperverdünnungsmittel) bei Raumtemperatur für 1 Std. </li><li> Counterstain den Zellkern, indem die Zellen mit 2 &amp; mgr; g / ml 4 abdeckt &#39;, 6-diamidino-2-phenylindole, waschen Sie die Zellen zweimal mit PBS für 15 Minuten, und montieren sie mit Anti-Fading-Montagelösung. </li><li> Erhalten Immunofluoreszenz Bilder bei den Anregungswellenlängen von 405 und 488 nm durch ein Laser-Scanning-konfokalen Mikroskop mit 20X Objektivvergrößerung verwendet. </li></ol> 2. Rat Korneaendothelium cryoinjury Modell und intrakamerale Injection <ol><li> Anästhesieren 12 Wochen alten männlichen Sprague-Dawley Ratten mit intramuskulären Injektionen von 2% Xylazin (5,6 mg / kg) und Tiletamin und Zolazepam (18 mg / kg). kneifen sanft die Haut der Tiere geeignete Anästhesie in Abwesenheit von Haut Zucken zu bestätigen. </li><li> Flößen Sie einen Tropfen von 0,5% Proparacainhydrochlorid dem rechten Auge jeder Ratte Augenschmerzen und Blinzelreflex zu minimieren. einflößenTetracyclin-Salbe auf das linke Auge kornealen Trockenheit zu verhindern. </li><li> Kühlen Sie eine Sonde aus rostfreiem Stahl (Durchmesser = 3 mm) in flüssigem Stickstoff. Tragen Sie die Sonde aus rostfreiem Stahl an die zentrale Hornhaut des rechten Auges für 30 Sekunden. Häufig PBS für das rechte Auge während des Verfahrens zu vermitteln kornealen Trockenheit zu verhindern. </li><li> Instill 0,1% Atropin und 0,3% Gentamicinsulfat unmittelbar nach cryoinjury und einmal täglich Augenschmerzen zu lindern von Ziliarspasmus ergeb und Infektion zu verhindern. <ol><li> Nach dem Eingriff halten die Ratten mit einer Heizlampe erwärmen und ihre Genesung alle 15 Minuten nach der Narkose zu beobachten, bis sie Motor wieder die Kontrolle. Zusätzlich Tetracyclin-Salbe auf das rechte Auge gelten für Hornhauttrockenheit während der Erholungsphase zu verhindern. </li></ol></li><li> Wiederholen Sie die Hornhaut cryoinjury für 3 aufeinander folgenden Tagen. </li><li> Für die Bereitstellung von Marimastat oder bFGF in die vordere Kammer des Rattenaugen, anästhesieren die Ratten, wie vorher beschrieben. Flößen Sie einen Tropfenvon 0,5% Proparacainhydrochlorid in das rechte Auge Augenschmerzen und Blinzelreflex zu minimieren. </li><li> Spülen Sie die Augenoberfläche mit sterilem PBS. Führen Vorderkammer Parazentese unter einem Operationsmikroskop mit einer 30 G-Nadel in eine 1 ml Spritze am paralimbal klare Hornhaut in einer Ebene oberhalb und parallel zu der Iris befestigt Einsetzen. </li><li> Drehen Sie die Nadel Schräge nach oben, und drücken Sie leicht die Hornhautwunde etwas wässrigen Humor und reduzieren den intraokularen Druck abzulassen. Injizieren 0,02 ml des intrakameral Droge. Vorsichtig komprimieren während der Nadel Rückzug der Nadel-Darm-Trakt mit einem Wattestäbchen. </li><li> Fotografieren Sie das äußere Auge bei einer angegebenen Zeitpunkt unter dem Operationsmikroskop. </li></ol> 3. Ernten der Ratte Corneal Knopf und Immunostaining <ol><li> Um die Ratten einschläfern, legen Sie sie in einer Euthanasie Kammer und ziehen lassen 100% CO 2 bei einer Füllrate von 10 bis 30% des Kammervolumens pro Minute. Pflegen CO 2 Infusion für eineweitere Minute nach mangelnder Atmung und verblasste Augenfarbe. </li><li> Dringen die Ratte Auge am Limbus mit einer scharfen Klinge. Schneiden Sie die Hornhaut mit Hornhaut-Schere entlang des Limbus. Flatten die Cornea auf einer Folie. Nehmen Sie zusätzliche radiale Einschnitte, wenn die Cornea einrollen. </li><li> Fix die Cornea mit 250 ul 4% Paraformaldehyd, pH 7,4, für 30 min bei Raumtemperatur. Permeabilisieren mit 250 ul 0,5% Triton X-100 für 5 min, und der Block mit 10% Rinderserumalbumin für 30 min. </li><li> Inkubieren der Kornea mit primären Antikörpern gegen ABC über Nacht bei 4 ° C mit einer Verdünnung von 1: 200 in Antikörperverdünnungsmittel. Waschen Sie die Cornea zweimal mit PBS für 15 min, und inkubieren sie mit dem sekundären Antikörper (1: 100 in Antikörperverdünnungsmittel) bei Raumtemperatur für 1 Std. Counterstain den Zellkern, indem die Zellen mit 2 &amp; mgr; g / ml 4 abdeckt &#39;, 6-Diamidino-2-phenylindol und die Zellen zweimal waschen mit PBS für 15 min. </li><li> Montieren Sie die Cornea in der Anti-Fading-Montagelösung. ObTain die Immunfluoreszenz-Bilder bei Anregungswellenlängen von 405 und 561 nm mit einem Laser-Scanning-konfokalen Mikroskop bei 20facher Objektivvergrößerung. </li></ol>

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The authors have no competing financial interests to declare.

CECs sind für ihre Neigung bekannt ENMT während der Zellproliferation zu unterziehen. Entwicklung von Strategien zur Unterdrückung des ENMT Prozess zu therapeutischen Zwecken, ein gründliches Verständnis der ENMT Mechanismus notwendig. Wir 2 beschriebenen Modelle ENMT, nämlich die Rinder-CEC in - vitro - Kulturmodell und Ratte Korneaendothelium cryoinjury Modell zu untersuchen. Unsere Ergebnisse zeigten die ENMT Prozess in beiden Modellen. Darüber hinaus wurde die ENMT Unterdrückungseffekt von Marimastat in beiden Modellen wiedergegeben werden, was darauf hindeutet, dass diese zwei Modelle denselben Mechanismus teilen. Die Rinder-CEC in - vitro - Kultur - Modell bietet eine einfache Manipulation. Zusätzlich, verglichen mit dem menschlichen oder Primaten - CEC in vitro Kulturmodellen in früheren Studien verwendet 17,18 sind Rinderaugen größer und leichter zu erfassen, und damit CECs zur Verfügung haben. In unserer Studie haben wir auf der entkernte Rinderaugen von einem örtlichen Schlachthof. Wegen der fres<html> h Natur der Rinderaugen, die genaue Zahl der Rinder CECs geerntet unterliegt Variation, bei etwa 1 x 10 5 Zellen pro Auge. Im Gegensatz dazu ist die Zahl der menschlichen CECs in einer normalen Hornhaut ungefähr 3 x 10 5 pro Auge. Diese Zahl ist niedriger in der Studie-Grade Cornea und ist sogar noch niedriger Resthornhautränder nach der Transplantation. Manipulationen während des Ernteverfahrens wird weiter die Anzahl der Zellen erhalten reduzieren. Darüber hinaus können Rinder LME vermehren und unterliegen ENMT spontan; Daher ist die bovine CEC in vitro Kulturmodell nützlich , die ENMT Mechanismus bei der Untersuchung und Screening ENMT Unterdrückungs Chemikalien, wie Marimastat. Obwohl es Bedenken bezüglich speziesbedingte Unterschiede, wie die proliferative Kapazität von CECs bleibt zeigte unsere Ergebnisse , dass die ENMT Signalweg von bovinem CECs der von menschlichem CECs ähnlich ist, einschließlich der Aktivierung der Wnt / β-Catenin 18,19. Zelt &quot;&gt; Während der Isolierung von Rinder-MEL, der Membran Descemet Peeling ist von entscheidender Bedeutung. In einigen Rinderaugen, der Membran Descemet enge Haftung mit dem Hornhautstroma haben kann, auf der geschälten Descemetschen Membran zu einer übermäßigen Stromagewebe führt. Weitere Trypsinierung führen kann die Freisetzung von Stromazellen Keratozyten, die in der Hornhaut Fibroblasten verwandeln, die die Zell Reinheit beeinflussen und damit die experimentellen Ergebnisse. in unserer Studie haben wir die Descemet-Membran sorgfältig unter einem Mikroskop abgezogen. weitere Zell Reinheit zu gewährleisten, kultiviert wir zunächst die geernteten Zellen in einem 6 cm-Schale, bis Zellkonfluenz. Nur wenn die Zellen in Form hexagonal waren, waren sie in weiteren Experimenten verwendet. Um die Ergebnisse der Rinder-CEC in - vitro - Kulturmodell untermauern, haben wir die Ratte Korneaendothelium cryoinjury Modell in früheren Studien angenommen 20,21. Nach cryoinjury unterzog sich die Ratte LME ENMT während der Regeneration, manifested durch die nukleäre Translokation von aktiven β-Catenin, was darauf hinweist, dass die Aktivierung der Wnt / β-Catenin-Signalweg zwischen den Arten bei CEC Regeneration konserviert ist. Darüber hinaus ist dieses Modell nützlich, um die Funktion von MEL untersuchen, weil niedrige CEC-Funktion offensichtlich Hornhautödems führt. In Kombination mit anderen Geräten, wie zum Beispiel Ultraschall biomicroscope, vordere Segment der optischen Kohärenztomographie und konfokaler Mikroskopie kann die Hornhautdicke quantitativ und damit CEC-Funktion widerspiegeln ausgewertet werden. Um die Regeneration von MEL stimulieren und zu unterdrücken ENMT nach Wundheilung, wir intrakamerale Injektionen von bFGF gefolgt von Marimastat Injektion verabreicht. Die Behandlung reduziert signifikant das Ausmaß der Hornhautödems. Frühere Studien haben die topische Anwendung von CEC wachstumsstimulierende Mittel, wie ROCK - Inhibitor und ENMT Unterdrückungsmittel, wie Notch - Inhibitor 21,22 verwendet. Allerdings lag das Eindringen von Drogen in die CECer wird durch das Hornhautepithel und Stroma beschränkt, die die Wirksamkeit der Behandlung 23 beeinflussen können. Im Gegensatz zur topischen Anwendung ermöglicht intracameral Injektions direkten Zugang zum Hornhautendothel durch Medikamente direkt in die vordere Kammer eingeführt wird. Daher kann die wachstumsstimulierenden und ENMT-unterdrückende Wirkung von Chemikalien unbiasedly cryoinjury folgenden werden bewertet. In unserer Studie führten wir Parazentese Vorderkammer des intraokularen Drucks vor intracameral Injektion zu senken. Ohne Parazentese verursacht eine abrupte Zunahme des intraokularen Drucks Hornhautödem oder sogar Prolaps der Iris durch die Nadelwege. Sanft Komprimieren der Darm-Trakt während Nadel Rückzug Hilfen die Wunde und verhindert intraokularen Infektion in abdichten.

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">trypsin</td>
			<td style="width:269px;">ThermoFisher Scientific</td>
			<td style="width:143px;">12604-013</td>
			<td style="width:340px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium and Ham&#39;s F12 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human epidermal growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insulin, transferrin, selenium&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholera toxin</td>
			<td>Sigma</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentamicin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amphotericin B</td>
			<td>ThermoFisher Scientific</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>111219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">marimastat</td>
			<td>Sigma</td>
			<td>M2699-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-active beta-catenin antibody</td>
			<td>Millpore</td>
			<td>05-665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-snail antibody</td>
			<td>Santa cruz</td>
			<td>sc28199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-slug antibody</td>
			<td>Santa cruz</td>
			<td>sc15391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>for staining of ABC of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11003</td>
			<td>for staining of ABC of rat corneal endothelium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>for staining of snail and slug of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibody diluent</td>
			<td>Genemed Biotechnologies</td>
			<td>10-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>ZEISS</td>
			<td>LSM510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine&#160;</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tiletamine plus zolazepam</td>
			<td>Virbac</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">proparacaine hydrochloride ophthalmic solution</td>
			<td>Alcon</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% atropine</td>
			<td>Wu-Fu Laboratories Co., Ltd</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3% gentamicin sulfate</td>
			<td>Sinphar Group</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">basic fibroblast growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>clinical drug&#160;</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Nach der Isolierung von Rinder-MEL wurden die Zellen in vitro kultiviert. Abbildung 1 zeigt die Phasenkontrastbilder der Rinder-MEL. Die hexagonale Form der Zellen bei Konfluenz angedeutet , dass die Zellen durch Fibroblasten während der Zellisolierung Hornhaut Stroma nicht kontaminiert wurden. Abbildung 2 zeigt die Immunfärbung , die Antikörper gegen ABC, Schnecke und Schnecke mit in einem angegebenen Zeitpunkt durchgeführt wurde. Abgesehen von phänotypischen Veränderungen in der in vitro Kultur, eine entsprechende nukleäre Translokation von ABC und EMT Regulatoren beobachtet. 3 veranschaulicht die Wirkung von Marimastat, eine Breitspektrum - MMP - Inhibitors auf der ENMT Prozess der in vitro kultivierten bovinen CECs. Figure 4 umfaßt die äußeren Augen Fotografien von Ratten nach cryoinjury durch intracameral Injektion folgte. 5 zeigt die Immunfärbung des r<html> auf der Hornhaut-Taste ausgestattet, wurde unter Verwendung von Antikörpern gegen ABC nach cryoinjury gefolgt von intrakamerale Injektion durchgeführt. Die nukleäre Translokation von ABC wurde in der PBS-Gruppe beobachtet und wurde in der bFGF-Gruppe signifikant erhöht, was auf die Aktivierung der Wnt / β-Catenin-Signalisierung und ENMT Prozess. Nach intracameral Injektion von bFGF durch Marimastat Injektion gefolgt wurde die Kernfärbung der ABC vermindert, was auf die ENMT hemmende Wirkung von Marimastat. <img alt="Abbildung 1" src="/files/ftp_upload/54329/54329fig1.jpg" /> Abb . 1: Phasenkontrast - Bilder von in vitro gezüchteten Rinder-MEL Nachdem auf der Kulturplatte ausgesät werden, die Rinder-MEL zunächst erschien Fibroblasten wie an den Tagen 3 und 6. Sie wurden mehr hexagonal bei Erreichen der vollständigen Konfluenz am Tag 9. Maßstabsbalken = 50 um. Repräsentative Bilder von 3 Replikaten.4329fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte hier klicken, um eine größere Version dieser Figur zu sehen. <img alt="Figur 2" src="/files/ftp_upload/54329/54329fig2.jpg" /> Abbildung 2: Immunfärbung von in vitro gezüchteten Rinder-MEL Während der in - vitro - Kultur des Rinder LME, die nukleäre Translokation von ABC, Schnecke und Schnecken wurde durch Tag erkannt 14. ABC:. Aktive β-Catenin. Maßstabsbalken = 100 &amp; mgr; m. Repräsentative Bilder von 3 Replikaten. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54329/54329fig3.jpg" /> Abbildung 3:. Immunostaining von in vitro gezüchteten Rinder-MEL mit oder ohne Marimastat bei verschiedenen Zell Einmündung Ebenen Immunostaining Dämonenschaulicht, dass ABC, Schnecke und Schnecke im Kern der Rinder-MEL offensichtlich waren 3. mit oder ohne 10 &amp; mgr; M Marimastat am Tag Marimastat jedoch deutlich reduziert die Kernfärbung von ABC, Schnecke und Schnecke am Tag 9, wenn die Rinder-MEL wurde vollständig konfluent. Maßstabsbalken = 100 &amp; mgr; m. Repräsentative Bilder von 3 Replikaten. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig3large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 4" src="/files/ftp_upload/54329/54329fig4.jpg" /> Abbildung 4:. Externe Auge Fotos von Ratten zu den angegebenen Zeitpunkten nach der cryoinjury Nach cryoinjury für drei aufeinander folgenden Tagen wurden die Ratten zu intrakamerale Injektion von 0,02 ml PBS oder 50 ng / ml bFGF am 3. Am Tag 6 Tag ausgesetzt 0,02 ml 10 &amp; mgr; M Marimastat wurde intrakameral in der bFGF / Mari Gruppe injiziert, während PBS in den anderen 2 Grou injiziert wurdeps (n = 9 in jeder Gruppe). Externe Auge Fotografien ergab Hornhautödems nach bFGF Injektion reduziert, während Marimastat weiter reduziert Hornhautödems verglichen mit bFGF allein. N = 9 in jeder Gruppe. Maßstabsbalken = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig4large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 5" src="/files/ftp_upload/54329/54329fig5.jpg" /> Abbildung 5:. Immunostaining von Rattenhornhautscheibchen Immunostaining der Ratte Hornhautscheibchen, die am Tag geerntet wurden 9 ergab wenig Kernfärbung von ABC in der PBS - Gruppe. In der bFGF-Gruppe gab es umfangreiche Kernfärbung von ABC, die maßgeblich an der bFGF / Mari-Gruppe reduziert wurde. Maßstabsbalken = 100 &amp; mgr; m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig5large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig5large.jpg" target="_blank">eine größere Version von thi zu sehen</a>Wert.

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Eine Primärkultur von Rinder Endothelzellen wurde verwendet, um den Mechanismus der Cornea-Endothelzellen-mesenchymale Transition zu untersuchen. Weiterhin wurde eine Ratte Hornhautendothel cryoinjury Modell kornealen Endothel-mesenchymale Transition in vivo zu demonstrieren.

In vitro und in vivo Modelle Corneal Endothelial-mesenchymale Transition zu studieren

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K Aufrufe.  Far Eastern Memorial Hospital.  Eine Primärkultur von Rinder Endothelzellen wurde verwendet, um den Mechanismus der Cornea-Endothelzellen-mesenchymale Transition zu untersuchen. Weiterhin wurde eine Ratte Hornhautendothel cryoinjury Modell kornealen Endothel-mesenchymale Transition in vivo zu demonstrieren. 

Serie: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Charakterisierung von Thymic Einschwingzeit Vorläufern in der Mouse Embryo Verwendung<em&gt; In Vivo</em&gt; Und<em&gt; In-vitro-</em&gt; Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Forschung

Entwicklungsbiologie

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

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

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

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

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

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

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Entwicklung eines<em&gt; In Vitro</em&gt; Assay Kontraktionsfunktion von mesenchymalen Zellen, die Unterzog Epithelial-mesenchymale Transition zur Bewertung

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Live Imaging der Maus Sekundärer Gaumen Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissektion, Immunofluorescent Färbung der Pilz Körper und Photorezeptor Neuronen im Gehirn Erwachsener Taufliege melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Anwendung von RNAi und Hitze-Schock-induzierte Transkription Faktor Ausdruck zu programmieren Keimzellen Neuronen in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualisierung der Knoten und Notochordal Platte In Gastrulating Maus-Embryonen mittels Rasterelektronenmikroskopie und ganze Mount Immunfluoreszenz

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Erwachsenen Zebrafisch Verletzungen Modelle zur Untersuchung der Auswirkungen von Prednisolon bei der Regeneration von Knochengewebe

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Ein In-vitro- Modell eines Systems Parallel-Platte Perfusion, bakteriellen Einhaltung der Gewebe Transplantat zu studieren

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Eine In-Vivo -Methode, um die Maus Blut-Hoden-Schranke Integrität zu studieren

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...