Research reported in this publication was supported by a generous gift of 14-month-old female CBA mice from the National Institute on Aging, Aged Rodent Colony (Nancy Nadon) and by Utah State University, School of Veterinary Medicine, Department of Animal, Dairy and Veterinary Sciences.

All methods described here were developed under National Research Council guidelines found in the Guide for the Care and Use of Laboratory Animals and have been approved by the Utah State University Institutional Animal Care and Use Committee.
NOTE: Ovarian transplantation procedures may involve mice (CBA/J strain) from 21 days of age to 18 months of age. Mice of the CBA/J strain become reproductively competent between 45 and 60 days of age and reproductively senescent between 10 and 12 months of age.
1. Transplantation of intact ovaries to aged mice
NOTE: This first protocol describes the procedure used during the transfer of ovaries from a young mouse (60 days of age) to a postreproductive female (12 months of age) of the same inbred strain. The same procedure is used for ovarian transplantation between any two female mice.
<ol>
	<li>Weigh and anesthetize the donor animal by intraperitoneal (I.P., 27 ga needle) injection, for example using a cocktail of ketamine, xylazine and acepromazine (65 mg/kg ketamine, 13 mg/kg xylazine 2.0 mg/kg, acepromazine; cocktail dose - 6.5 ml/kg). Return the animal to the heated home cage until the anesthesia has taken effect.</li>
	<li>Once anesthetized, remove the animal from the home cage and place on heated paper towels. Using clippers (#40 blade preferred), remove the hair a few mm lateral to midline on each side, starting just below the ribs and moving distally, preparing a clipped &#39;patch&#39; approximately 2-3 cm square on either side of the prone mouse leaving a strip of hair approximately 1-2 cm covering the midline.</li>
	<li>Lightly wipe the clipped areas with 70% ethanol and, apply betadine solution to the clipped site with a cotton-tipped swab.</li>
	<li>Apply antibiotic gel for eye protection and administer meloxicam (2.0 mg/kg) subcutaneously (S.C., 27 ga needle) on the lateral aspect of the animal&#39;s neck for postoperative pain relief.</li>
	<li>Place the animal in prone position on a heated sterile surgical field, and wipe the clipped area with a 70% ethanol-soaked 2 cm x 2 cm cotton gauze.</li>
	<li>Make a 1.0-1.5 cm paralumbar incision and bluntly dissect the skin from the underlying fascia. Make another incision through the fascia. Bluntly dissect laterally under this fascia as superficially as possible until reaching the abdominal cavity</li>
	<li>Place a small surgical drape over the incision (cut from 2 cm x 2 cm gauze) and wet the drape with sterile saline.</li>
	<li>Using small forceps, locate the adipose tissue that surrounds the ovary in the abdominal cavity and gently extract the fat pad and ovary and place it on the wetted gauze drape. The ovarian fat pad will appear more &#39;white&#39; than the surrounding adipose tissue.</li>
	<li>Place a 1.5 inch Bulldog clamp on the fat pad (taking care not to obstruct the ovarian bursa) to hold the tract in place during the procedure.</li>
	<li>Move the dissecting microscope into place and clearly identify the ovarian bursa. Grasp the bursa with two watchmaker forceps and incise the ovarian bursa opposite the ovarian hilum to expose the ovary. If the intended access area of the bursa is highly vascularized, apply a drop of epinephrine (1:100,000) with a syringe to the bursal surface to reduce bleeding at the time of tearing.</li>
	<li>Gently remove the ovary from the ovarian bursa and excise the ovary by clamping at the ovarian hilum with a watchmaker forceps to prevent bleeding.</li>
	<li>Place the donor ovary in watch glass filled with 4&#176; C sterile saline, loosely cover and set aside until ready for transplantation. Ideally, place the watch glass under an additional dissecting microscope so that the ovaries can be easily grasped by the surgeon at the time of transplantation.</li>
	<li>At this point, either euthanize or set aside the donor mouse&#160;(must be continuously monitored) to receive a transplanted ovary, tissue, cells or a placeholder glass bead at a later time point.</li>
	<li>With a clean surgical field and instruments, ovariectomize the recipient animal as above. Older mice tend to have an increased amount of adipose tissue and may require an increased anesthetic dose (up to 20%).</li>
	<li>After excision of the ovary from the recipient, place a small piece of hemostatic foam pad inside the empty bursa to control bleeding and aid in clotting and place a small piece of saline-soaked gauze over the exposed site.</li>
	<li>Locate a donor ovary (or tissue, cells, placeholder glass bead) with a watchmaker forceps in one hand and move into the microscope field-of-view. With another forceps in the opposite hand, remove the gauze and simultaneously remove the hemostatic foam pad from the bursa while placing the new ovary inside the bursa.</li>
	<li>If the new ovary does not fit inside the recipient bursa, cut the donor ovary in half (or 1/3 or 1/4) and place into the bursa in pieces. If the bursa does not completely cover the entire ovary, place a suture through the bursa, through the ovary and back through the bursa to hold the ovary in place (see Discussion section). 
	NOTE: The glass place-holder beads can be difficult to handle with watchmaker&#39;s forceps.</li>
	<li>When placing a glass bead inside the bursa, pick up the bead with larger forceps and drop on top of the fat pad next to the ovarian bursa. Roll the bead into the bursa.</li>
	<li>Pick up a Castroviejo needle holder already loaded with a tapered needle and 9-0 or 10-0 monofilament suture, locate the torn edge of the bursa and close the bursa using one or two Halsted sutures. 
	NOTE: Suturing can alternatively be performed with watchmaker or other small forceps.</li>
	<li>Irrigate the site lightly with sterile saline, remove the fat-pad clamp and return the ovary/fat pad to the abdomen.</li>
	<li>Remove the drape and close the abdominal wall with 6-0 chromic gut or vicryl. This step can be done with or without the microscope.</li>
	<li>Move the microscope aside (if not moved in the previous step) and close the skin with 9 mm wound clips.</li>
	<li>Place the animal in a heated recovery cage shaded from light and monitor the animal continuously until recovery from anesthesia. Additional meloxicam may be used 24 hours post-operatively if indicated. Remove the wound clips approximately nine days post-operatively. 
	NOTE: No immunosuppressive techniques are employed and no evidence of graft-versus-host disease has been detected post-transplantation or at death.</li>
</ol>
2. Transplantation of germ cell depleted ovaries to intact mice.
NOTE: This protocol involves the transfer of ovaries from a young, germ cell-depleted mouse to a postreproductive female. In this protocol, the ovary recipients and the ovariectomy and transplantation procedures are as above with the exception that the transplanted ovaries have been chemically depleted of primordial and primary follicles/germ cells. Remaining secondary and antral follicles will undergo recruitment/atresia leaving the ovary devoid of germ cells. Germ cell depletion with 4-vinylcyclohexene diepoxide (VCD) is well described16. VCD is a toxin. Because the VCD-treated ovaries are being transplanted to a non-treated mouse, this procedure benefits from a VCD-treated ovary in a mouse not exposed to the toxic effects of VCD. This protocol describes the procedure for germ cell-depletion in young, ovary-donor mice.
<ol>
	<li>Dilute VCD (64 mg/mL) in sesame oil for injection and store at 4&#176; C until use.</li>
	<li>Inject ovary donors once a day with VCD I.P. (160 mg/kg) for 15 days beginning at 28 days of age. Inject control mice with sesame oil following the same schedule.</li>
	<li>At 50 days of age, check VCD-treated and oil-only-treated mice for cessation of reproductive cyclicity for 10 days as above. At 60 days of age, VCD-treated mice should display no cytological evidence of gonadal input.</li>
	<li>Recover ovaries from 60-day-old mice and transplant as above.</li>
	<li>Determine successful transplantation of germ cell-depleted ovaries by detection of a lack of reproductive cycling and increased levels of circulating androstenedione. Androgen:Estrogen ratios increase significantly after germ cell depletion with VCD17.</li>
	<li>Initiate vaginal cytology 10-14 days postoperatively and collect data for at least 10 consecutive days to ensure accurate determination of cyclicity.</li>
</ol>

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The authors have nothing to disclose.

Ovariectomy and transplantation of intact and germ cell-depleted ovaries have been conducted in mice ranging from 21 days to 18 months of age. When performing an ovariectomy where it is desirable to maintain the ovarian bursa intact, care must be taken when incising the bursal tissue. In mice older than six months of age, much of the bursa becomes overlaid with the fat pad, which limits the areas that can be used for access. In these cases, it is often advantageous to tear the bursa open close to the fat pad border. This...

The influence of reproduction on health and life span is often mistakenly thought of as restricted only to the period encompassing reproductive competency. While the lifestyle and phenotypes associated with the reproductively competent phase of the life span are easily differentiated from the pre- and postreproductive phases, reproductive status influences health throughout all phases of the life span. The period of reproductive competency is often referred to as the reproductive life span, differentiating itself from the chronological life span. This terminology lends itself to an artificial separation of the two life-span designations when in reality, both are closely tied to each other with events affecting one, more often than not, affecting the other as well.
Evidence over the past decade indicates that an individual&#39;s reproductive status is associated with an increased risk of developing chronic health conditions1. One study documented an association between shorter life spans and reproductive failure for a cohort of men2. The association is even more striking in women. Cardiovascular disease is rare in premenopausal women3, but ovarian failure increases cardiovascular disease sharply at menopause4 and in young women with premature ovarian failure5. Insulin resistance6 and bone loss increase at menopause7 and almost two-thirds of Americans with Alzheimer&#39;s disease are women8. Well-defined changes in ovarian signaling mark the transition from a premenopausal, disease-resistant state, to a postmenopausal, disease-burdened state. This stable transition, presents an opportunity to identify mechanisms that accelerate disease risks in aging.
Recently, transplantation of cryopreserved ovarian tissue has been used to restore fertility in women that undergo medical procedures which may endanger the germ line cells, such as chemotherapy. Transplantation of ovarian tissue can also be used to produce viable offspring in animals with non-ovarian based infertility, including transgenic mice9. Ovarian transplantation is an efficient method to separate the influence of reproductive function or reproductive aging from chronological aging per se.
The well-established supportive role for ovarian hormones in many aspects of female health implicates the loss of hormone production from actively cycling germ cells, as the principal cause of increased disease risks at menopause. While the value of ovarian hormones in female health is unquestionable, replacing these hormones in peri- and postmenopausal women has been unreliable in restoration of the health benefits enjoyed by young women with young ovaries. These health benefits are reliably restored by heterochronic transplantation of young ovaries to postreproductive female mice10,11,12,13,14.
Reproductive status of both donor and recipient mice should be determined prior to and after transplantation or ovariectomy procedures. Presumptive postreproductive mice that display signs of gonadal input prior to surgery at 12 months of age are excluded from these experiments. Gonadal input is determined by vaginal cytology. Data on vaginal cytology are collected for at least 10 consecutive days pre-surgery to ensure a postreproductive state in recipient mice and actively-cycling ovaries in donor mice. One estrous cycle is defined as the period from the day nucleated epithelial cells first appear (i.e., proestrus) to the day preceding the next appearance of nucleated epithelial cells in the vaginal smear, provided that there is a period of leukocytic presence (i.e., diestrus) in between. Estrus is determined by the presence of large, squamous epithelial cells, with or without nuclei. Mice that display no cyclic activity for a 10 day period before and/or after surgery are determined to have no gonadal input for said period. Mice that display at least one full estrous cycle in a 10 day period before and/or after surgery are determined to have gonadal input for said period. Female mice should be housed in the presence of male mice or exposed to soiled bedding from a male&#39;s cage to ensure consistent exposure to cycle-inducing stimuli.
These transplantation procedures are most efficiently accomplished with two surgeons working simultaneously on donor and recipient mice. However, the procedures are easily accomplished by one surgeon, with the donor ovaries most often recovered prior to beginning the ovariectomy procedure on the recipient mouse. This protocol describes the procedure for the single surgeon. Exposure of the reproductive tract and closure of the site is normally done without a microscope, whereas the removal and replacement of the ovaries is done under a microscope. As with learning any surgical procedure, incision sites may be larger and surgeries may take longer while first learning the procedure and will generally decrease with experience.

<table><tbody><tr><td>Dissecting microscope</td><td>Any dissecting scope with an adjustable working distance.</td><td></td><td></td></tr><tr><td>Warm Water Recirculator and appropriate pad for your size surgical/recovery field.</td><td>Stryker/Gaymar</td><td>TP700</td><td>Circulating water pads produce even and consistent temperature operating and recovery fields.</td></tr><tr><td>Surgical instruments-dissection instruments for access to the reproductive tract</td><td></td><td></td><td>Choice of specific surgical instruments is highly surgeon-specific</td></tr><tr><td>Surgical instruments-fine instruments for the transplantation procedure.</td><td></td><td></td><td>Choice of specific surgical instruments is highly surgeon-specific</td></tr><tr><td>Suture material for abdominal wall</td><td>eSutures</td><td>G3756K: 6-0 MILD CHROMIC GUT 18&quot; HE-7 CUTTING, DOUBLE ARMED</td><td></td></tr><tr><td>Suture material for ovarian bursa</td><td>eSutures</td><td>2809G: 9-0 or 2810G: 10-0 ETHILON BLACK 5&quot; BV130-5 TAPER</td><td>10-0 suture can be difficult to locate if dropped on the surgery field.</td></tr><tr><td>Absorbable gelatin sponge</td><td>PFIZER.</td><td>Gelfoam- PFIZER 09-0315-08.</td><td>cut into small (~2mm) pieces under sterile conditions.</td></tr><tr><td>Staples for skin closure</td><td>Becton Dickinson</td><td>MikRon Autoclip 9mm (BD 427631)</td><td></td></tr><tr><td>Eye ointment.</td><td>MWI Veterinary Supply Co.</td><td>Puralube sterile opthalmic ointment</td><td></td></tr><tr><td>Syringes for anesthesia, analgesia and epinephrine</td><td>MWI Veterinary Supply Co.</td><td>BD 305536</td><td>27g x 3/8in,Intradermal Bevel 1/2ml</td></tr><tr><td>Syringes for saline.</td><td>MWI Veterinary Supply Co.</td><td>BD 309657.</td><td>3ml syringe, luer-lok tip, 27g needle</td></tr><tr><td>Clippers/blade</td><td>Andis</td><td>Super AGR+&amp;trade; Cordless Detachable Blade Clipper and a CeramicEdge&amp;reg; #40 blade.</td><td>Any clippers with a fine blade.</td></tr><tr><td>Ethanol</td><td>University chemical supply</td><td></td><td>dilute to 70% for use.</td></tr><tr><td>Betadine solution</td><td>MWI Veterinary Supply Co.</td><td>003233</td><td></td></tr><tr><td>2x2 gauze pads</td><td>MWI Veterinary Supply Co.</td><td>003073</td><td>autoclaved with surgery packs</td></tr><tr><td>Borosilicate glass beads</td><td>Sigma-Aldrich Co.</td><td>Z273619</td><td>Autoclave in sintilation vials</td></tr><tr><td>4-vinylcyclohexene diepoxide</td><td>Sigma-Aldrich Co.</td><td>94956</td><td></td></tr><tr><td>Sesame oil</td><td>Sigma-Aldrich Co.</td><td>S3547</td><td></td></tr></tbody></table>

orthotopic ovarian transplantation procedures to investigate the life- and health-span influence of ovarian senescence in female mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Ovariectomy at 21 days of age will avoid major up-regulation of the reproductive system at the onset of puberty and eliminate other influences the female gonad might have in addition to direct effects of gonadal hormones. Reproductive decline in CBA/J mice usually begins with irregular cycles at 8-10 months of age. At 11 months of age, most females in this line of mice have reached a point of reproductive failure15 with a complete loss of responsive oocytes by 12 m...

Watch this Scientific Journal Video about Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice at JoVE.com

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

orthotopic-ovarian-transplantation-procedures-to-investigate-life

Research

JoVE Journal

Developmental Biology

10.8K Views.  Utah State University. Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Video: Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

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.

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 ...

Immunology and Infection

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

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

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

Behavior

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.

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

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.

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

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Medicine

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

Biology

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Ovarian Fat Pad Transplantation Assay: An In Vivo Technique to Introduce Cells into Ovarian Fat Pad in Mouse Models

Source: Flesken-Nikitin, A. et al. <a href="https://www.jove.com/t/54444">Transplantation Into the Mouse Ovarian Fat Pad.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an assay for transplantation of experimental cells and tissue fragments into the mouse ovarian fat pad. This method can be used to monitor cell regeneration and tumor transformation via non-invasive techniques.

Test przeszczepu poduszeczki tłuszczowej jajnika: technika in vivo wprowadzania komórek do poduszeczki tłuszczowej jajnika w modelach mysich

Source: Flesken-Nikitin, A. et al. Transplantation Into the Mouse Ovarian Fat Pad. J. Vis. Exp. (2016)
In this video, we demonstrate an assay for ...

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

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

Source: Koya, Y. et al. <a href="https://www.jove.com/t/54353">Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells.</a> J. Vis. Exp. (2016)
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.

Ortotopowa inokulacja komórek raka jajnika do mysiego jajnika: procedura mająca na celu stworzenie mysiego modelu raka jajnika do badania rozsiewu komórek w otrzewnie

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

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

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Assessment of Sexual Behavior of Male Mice

Accurate Follicle Enumeration in Adult Mouse Ovaries

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

Transplantation Into the Mouse Ovarian Fat Pad

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

Test przeszczepu poduszeczki tłuszczowej jajnika: technika in vivo wprowadzania komórek do poduszeczki tłuszczowej jajnika w modelach mysich

Ortotopowa inokulacja komórek raka jajnika do mysiego jajnika: procedura mająca na celu stworzenie mysiego modelu raka jajnika do badania rozsiewu komórek w otrzewnie