עבודה זו מומנה על ידי מענקים CIRM # TR-1250, RT1-01108, ו CL1-00502.

הערה: כל הנהלים בעלי חיים חייב להיות מאושר על ידי IACUC או שווה ערך. כל הציוד הכירורגי יש לעקר לפני הניתוח. כפפות סטריליות, וילונות גזה יש להשתמש. 1. הכנה לפני הניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג 6 בן שבוע מ.א.ס. מאסכאלאס CbySmn.CB17-Prkdc SCID / י עכברים ממין זכר או זן immunocompromised השני של העכבר. </li><li style=";text-align:right;direction:rtl"> לעקר את כל מכשירי ניתוח, כפפות, ו גזה. </li><li style=";text-align:right;direction:rtl"> לנתק hPSCs להיות מוזרק עם accutase. </li><li style=";text-align:right;direction:rtl"> ספירת תאים ו resuspend 1,000,000 תאים לכל μL 20-30 ב matrigel בדילול 01:01 ב DMEM/F-12. שמור על תאים על הקרח עד גמר. </li><li style=";text-align:right;direction:rtl"> שים לב כי לעיתים קרובות כדאי לעשות ריצה דרך של ניסוי שבו Trypan כחול מוזרק. זה יעזור לזהות בעיות פוטנציאליות לפני כל התאים מבוזבז. </li><li style=";text-align:right;direction:rtl"> ב ביבר, להרדים את העכבר בהתאם לנהלים המקובלים במוסד שלך. בניסויים שלנו, השתמשנו anesthesia מכונה עם isoflurane. העכברים הוכנסו בתחילה עם חמצן 1L/min ו 3-4% isoflurane בתא אינדוקציה. הרדים פעם, חרוט האף עם חמצן 1L/min ו 2-3% isoflurane היה בשימוש. </li><li style=";text-align:right;direction:rtl"> לגלח את הבטן עם קוצץ, ולנקות את הקיר הקדמי של הבטן העכבר, החל ממרכז הבטן ועבודה עם כיוון השעון כלפי חוץ. הפתרון הראשון Povidone-יוד השימוש מכן לשטוף עם אתנול 70%. לחזור 3 פעמים, שינוי דגימות בכל פעם. להעביר את בעל החיים משטח חימום כדי לשמור על בעל החיים חם בתוך התרבות רקמות או מכסה המנוע לנתח. </li><li style=";text-align:right;direction:rtl"> לעשות חתך אורכי 1 ס&quot;מ דרך העור הצפק עם מספריים כירורגיות סטריליות ממש מתחת לרמה של הירך משותפת. </li><li style=";text-align:right;direction:rtl"> תוך כדי לחיצה הצפק עם מלקחיים, להגיע לעבר הירך הימנית עם forcept אחר סטרילית ולשלוף את רקמת שומן לבן יחד עם האשכים המצורפים. </li><li style=";text-align:right;direction:rtl"> מניחים את האשכים על גזה סטרילי. </li><li style=";text-align:right;direction:rtl"> מלא טוברקולין או המילטון (1cc) מזרק Wiה hPSCs כדי להיות מוזרק. שים לב, זה רעיון טוב לכלול פס בקרה hPSC שאתה יודע שהוא pluripotent כגון WA09 (הידוע גם בשם H9). בדרך זו יש לך ביקורת חיובית שאפשר להשתמש בו כדי לקבוע אם הזריקה שלך או ניתוח היה פגום. </li><li style=";text-align:right;direction:rtl"> לאט לאט להזריק את hPSCs (20-30 μL) למרכז הקפסולה testis הרחק מכל כלי הדם הגדולים, לעצור אם הקפסולה האשכים מתחיל להתנפח. </li><li style=";text-align:right;direction:rtl"> להוציא את המחט לאט לאט כדי למנוע רפלוקס של התאים. </li><li style=";text-align:right;direction:rtl"> באמצעות מלקחיים, להעביר את האשכים והגב רקמת שומן למקומו המקורי בבטן. </li><li style=";text-align:right;direction:rtl"> לסגור את הצפק עם 2 או 3 תפרים reabsorbable ולסגור את העור עם autoclips. </li><li style=";text-align:right;direction:rtl"> העכבר יש לשמור חם עד שהוא מתאושש וקיבל צורה כלשהי של כאבים (ראה מה שמקובל ביבר שלך) לאחר הניתוח פעמיים ביום במשך 1-2 ימים. שים לב, לא הרדמה או משכך כאבים עם להפריע להתפתחות הגידול. </li><li style=";text-align:right;direction:rtl"> צג הדואר חיה על הגידול שבועות 6-12. לעתים נדירות ביותר, גידולים עשוי לגדול ל 5 מ&quot;מ בגודל קודמות עד 6 שבועות לאחר ההזרקה, ולכן חשוב לעקוב אחר בעלי החיים. אם זה יקרה, בעלי החיים צריך להיות מורדמים וגידולים את עיבוד וניתוח כרגיל. </li><li style=";text-align:right;direction:rtl"> לאחר הגידול הוא מוחשי ומגיעה בגודל 5 מ&quot;מ בערך, להרדים את העכבר ולהקריב אותו על פי הנהלים המקובלים פרוטוקול של בעלי חיים. </li><li style=";text-align:right;direction:rtl"> הסרה של הגידול המסמך בהתאם - גודל, מדד צילום, לשקול. </li><li style=";text-align:right;direction:rtl"> גזור הגידול לחתיכות קטנות ולתקן בפתרון paraformaldehyde 4%. חנות paraformaldehyde 4% עד דגימות נשלחות פתולוג עבור מכתים, חתך וניתוח. </li></ol> 2. נציג תוצאות: כאשר בפרוטוקול זה נעשה כפי שתואר לבין הקו התא שהוחדר הוא pluripotent, מוחשי, ברור מבחינה חזותית, הגידול צריך להקים בתוך 12 שבועות לכל היותר. עבור קווי hPSC שהוקמו כמו WA09, אנו בדרך כלל רואיםגידולים בתוך 6 שבועות. עבור קווי iPSC אין זה יוצא דופן לראות גידולים בתוך 8-10 שבועות. חשוב מאוד להכניס עכברים עם קו זה ידוע pluripotent, כביקורת חיובית, על מנת להיות בטוח כי ההליך בוצע בצורה נכונה. גידולים בדרך כלל נראים הטרוגנית מאוד ויש להם ציסטות מצורפים רבים (איור 1). ניתוח של דגימות הגידול על ידי פתולוג צריך להראות רקמות הבדיל מכל שלוש שכבות הנבט (איור 2). <img alt="איור 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> באיור 1. אופיינית תפלצת רוחנית נגזר hPSC בכמוסה testis. בעקבות הפרוטוקול המתואר, WA09 מיליון תאים הוזרקו הקפסולה testis של העכבר נפגעת מערכת החיסון. שישה שבועות לאחר מכן נצפתה תפלצת רוחנית. א) משך תפלצת רוחנית של העכבר. ב) תקריב תמונה של תפלצת רוחנית. הערה המבנים ואת ההטרוגניות ציסטה. <img alt="איור 2" src = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; איור 2. Hematoxylin וחתכים Eosin מוכתמים מן תפלצת רוחנית להראות רקמות של כל שכבת נבט. לאחר קיבוע, תפלצת רוחנית נותח ומוכתמים Hematoxylin ו Eosin. ניתוח על ידי פתולוג חשף את נוכחותם של תאים כל אחד 3 שכבות הנבט.

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אין ניגוד עניינים הצהיר.

השיטה המוצגת כאן מספקת אמינה מאוד, באמצעים פשוטים של יצירת teratomas מ hPSCs הקפסולה testis. ישנם מספר פרמטרים קריטיים בטכניקה זו. בפרט, חשוב להזריק שורות תאים hPSC שידוע כי הם להיות pluripotent כביקורת. פרמטרים חשובים נוספים כוללים את מרווח הזמן בין הזרקה והתבוננות הגידול. על שורות תאים כמו WA09, teratomas צריך לשמור על 6-8 שבועות. על קווים חדשים iPSC אנו מוצאים כי לעתים קרובות 10 שבועות נדרשים. חשש נוסף הוא מספר התאים המוזרקים. אנחנו מזריקים מיליון תאים, אך assay יכול להיעשות בקלות עם פחות תאים. בנוסף, בינוני זריקה חשובה. אנו מוצאים כי אנחנו מקבלים את התוצאות הטובות ביותר כאשר התאים מוזרקים בתערובת 01:01 של matrigel ו DMEM/F-12, לעומת PBS או DMEM/F-12.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> Accutase </td><td> Invitrogen </td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 113300-032 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

בתאי גזע pluripotent (PSCs) יש מאפיין ייחודי כי הם יכולים להתמיין לתאים מכל שלוש שכבות הנבט. זה גורם להם להיות כלי רב ערך פוטנציאלית לטיפול במחלות שונות. עם כניסתו של המושרה בתאי גזע pluripotent (iPSCs) ומחקר המשך בתאי גזע עובריים אנושיים (hESCs) יש צורך מבחני זה יכול להוכיח כי קו מסוים הוא תא pluripotent. שידור germline כבר את תקן הזהב עבור הוכחת pluripotence של תא גזע עובריים בעכבר (המסקלין) שורות 1,2,3. שימוש assay זה, החוקרים יכולים להראות כי הקו המסקלין יכול לעשות את כל סוגי התאים בעובר כולל בתאי הנבט 4. עם הדור של האדם ESC קווים 5,6, assay המתאים להוכיח pluripotence של תאים אלה לא היה ברור מאז ESCs אדם לא יכול להיבחן לשידור germline. כתחליף, assay תפלצת רוחנית משמשת כיום להפגין pluripotency בתאי גזע האדם גזע pluripotent (hPSCs) 7,8,9. למרות assay זה הגיע לאחרונה תחת בדיקה וטכנולוגיות חדשות נבדקות באופן פעיל, assay תפלצת רוחנית הוא תקן הזהב הנוכחי 7. ב assay זה, התאים המדוברים מוזרקים לעכבר נפגעת מערכת החיסון. אם התאים pluripotent, תפלצת רוחנית בסופו של דבר לפתח קטעי הגידול יראה רקמות מכל 3 שכבות הנבט 10. ב assay תפלצת רוחנית, hPSCs יהיה ניתן להזריק לתוך אזורים שונים של העכבר. האתרים הנפוצים ביותר כוללים הזרקת testis הקפסולה, הקפסולה כליות, כבד, או את הרגל או מתחת לעור או לשריר 11. כאן אנו מתארים את פרוטוקול חזקים עבור הדור של teratomas מ hPSCs באמצעות הקפסולה testis כאתר של הגידול.

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

אדם בתאי גזע pluripotent (hPSCs) יש פוטנציאל לטפל מספר עצום של מחלות שונות. השירות של תאים אלה הוא בכך שהם יכולים להתמיין לכל סוג תא בגוף. כאן אנו מתארים את assay תפלצת רוחנית, אשר משמש כדי להדגים את pluripotence של hPSCs.

תפלצת רוחנית דור הקפסולה testis

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

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20.7K Views.  Scripps Research Institute. אדם בתאי גזע pluripotent (hPSCs) יש פוטנציאל לטפל מספר עצום של מחלות שונות. השירות של תאים אלה הוא בכך שהם יכולים להתמיין לכל סוג תא בגוף. כאן אנו מתארים את assay תפלצת רוחנית, אשר משמש כדי להדגים את pluripotence של hPSCs.

Video: Teratoma Generation in the Testis Capsule

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

היווצרות של האפיתל ערמונית אנושי באמצעות רקומבינציה של רקמות ואברי מין מכרסם mesenchyme סינוס ותאי גזע אנושיים

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

תאי בידוד של תאי Sertoli ו Peritubular מן חולדת האשכים

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Immunology and Infection

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

הכנת מדרוזופילה זחל ופופל אשכים לניתוח חטיבת תאים בשידור חי, רקמה שלמה

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Developmental Biology

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

אקסטרה סלולר מבוסס מטריצה ודור נוסף ללא מטריקס סלולרי של אורגנואידים אשכים Murine

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

נבט השתלת ורקמות testis Xenografting ב עכברים

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Biology

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

הדור של מבנה האשך של פורצין עם הארכיטקטורה הספציפית ביותר באמצעות התרבות של המיקרוגל

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

אקס תרבות Vivo של דרוזופילה גלמי אשך וציסטות נבט אונליין רווק בן: Dissection, הדמיה, ותרופתי טיפול

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Renal Capsule Tumor Xenografting: A Technique to Generate an Experimental Animal Model to Evaluate Prostate Carcinogenesis

Source: Nicholson, T. M. et al. <a href="https://www.jove.com/video/50574" target="_blank">Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia.</a> J. Vis. Exp. (2013)
In this video, we demonstrate the implantation of human-derived prostate cancer tissue grafts into the renal capsule of an immunocompromised recipient mouse. This technique can be used to assess tumor progression and develop novel therapeutic strategies.

גידול בקפסולה כליה Xenografting: טכניקה ליצירת מודל ניסיוני של בעלי חיים להערכת סרטן הערמונית

Source: Nicholson, T. M. et al. Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign ...

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Animal model studies

Spermatogenesis

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of seminiferous tubules. These spermatogonial stem cells divide asymmetrically to give rise to additional stem cells (meaning that these structures &ldquo;self-renew&rdquo;), as well as sperm progenitors, called spermatocytes. Importantly, this method of asymmetric mitotic division maintains a population of spermatogonial stem cells in the male reproductive tract, ensuring that sperm will continue to be produced throughout a man&rsquo;s lifespan. As spermatogenesis proceeds, spermatocytes embark on meiosis, and each ultimately divides to form four sperm&mdash;each with only 23 chromosomes&mdash; that are expelled into the male reproductive tract. Interestingly, this is in contrast to oogenesis in women, during which only a single egg is generated for every progenitor cell.

<h4>Spermatogenesis and Capacitation</h4>

At the end of spermatogenesis, sperm demonstrate their characteristic shape: a &ldquo;head&rdquo; harboring minimal cytoplasm and a highly condensed nucleus, as well as a motile tail (flagellum). They are small cells, with no organelles such as ribosomes, ER or Golgi, but do have many mitochondria around the flagellum for power. Just below the head is the acrosomal vesicle which contains hydrolytic enzymes to penetrate the egg outer coat&mdash;the contents are released by exocytosis.

Sperm cells endure an additional maturation process after spermatogenesis termed capacitation. Interestingly, capacitation only occurs in sperm once these cells have been introduced into the female genital tract. Here, components of the female environment&mdash;like mucus&mdash;elicit critical changes in these gametes, among them the removal of cholesterol from their membranes. This enables different molecules to enter into sperm, such as bicarbonate ions, which can activate new proteins and increase tail movements. Such rigorous motions help sperm to navigate towards, and ultimately infiltrate, the thick protein-based zona pellucida surrounding an egg cell. In addition, cell membrane alterations lead to an increase in the concentration of calcium ions in sperm, which is also necessary for zona pellucida penetration. Thus mature sperm, demonstrating rapid tail activity and the ability to locate, bind and penetrate an egg, require the unique conditions of the female reproductive organs.

<h4>Factors that Affect Spermatogenesis</h4>

Several factors can affect sperm production. One well-documented influence is exposure to heat in the scrotum: men shown to sit for long periods of time (like cab drivers) or metal welders demonstrate increased risk of infertility or sperm with decreased motility. In addition to temperature, certain drugs have also been determined to have adverse effects on spermatogenesis. These include hormone antagonists used to address prostate conditions, cancer treatments, and even specific antibiotics. As a result, some professionals are emphasizing the need to warn men of the effects of these drugs on their reproductive health, rather than only focusing on what medications a woman planning a pregnancy should avoid.

Spermatogenesis and Capacitation

ספרמטוגנסיס (יצירת תא זרע)ֿ

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of ...

Education

JoVE Core

Human Biology

Reproduction and Development

KEY TERMS AND CONCEPTS

Testes: Gross Anatomy

The testes, also known as testicles, are the male gonads. They are housed within the scrotum, a sac-like structure located beneath the penis. The scrotum&#39;s primary role is to regulate the temperature of the testes, which is crucial for sperm production.
Each testis is surrounded by the tunica albuginea, a dense connective tissue layer that provides structural support and protection. This layer is covered by an outer serous membrane called the tunica vaginalis, which helps reduce friction between the testes and surrounding structures.
The scrotum, composed of a thin layer of skin, superficial fascia, and muscle layers, provides some physical protection. It is divided into two chambers by the scrotal septum, a fibrous partition, each housing one testis. The dartos muscle in the subcutaneous layer wrinkles the scrotal skin to reduce heat loss. In contrast, the cremaster muscles, made of skeletal muscle fibers, draw the testes closer to the body in response to cold temperatures or during sexual arousal. These thermoregulatory functions help maintain an optimal environment for the testes to function effectively.
The paired testes produce sperm and hormones essential for reproduction, and their optimal functioning is necessary for male fertility.

אשכים: אנטומיה גסה

The paired testes are the male gonads.
Each testis, weighing 10 to 15 grams, measures about 5 cm long, 3 cm wide, and 2.5 cm thick.
The tunica albuginea, ...