Name: orthotopic ovarian transplantation procedures to investigate the life- and health-span influence of ovarian senescence in female mice
Uploaded: 2018-02-12T13:00:00
Description: 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

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...

<ol>
	<li>National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Fertility and Infertility Branch. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Fertility+Status+as+a+Marker+of+Overall+Health'.+"Support+studies+that+investigate+fertility+status+as+a+marker+of+overall+health+for+both+men+and+women".">'Fertility Status as a Marker of Overall Health'. "Support studies that investigate fertility status as a marker of overall health for both men and women".</a> New Research Priorities. , (2016).</li><li>Eisenberg, M. L., Li, S. F., Behr, B., Cullen, M. R., Galusha, D., Lamb, D. J., Lipshultz, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semen+quality,+infertility+and+mortality+in+the+USA.">Semen quality, infertility and mortality in the USA.</a> Hum. Reprod. 29 (7), 1567-1574 (2014).</li><li>Thom, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.+Heart+disease+and+stroke+statistics+-+2006+update+-+A+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics - 2006 update - A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 113 (6), E85-E151 (2006).</li><li>Shuster, L. T., Rhodes, D. J., Gostout, B. S., Grossardt, B. R., Rocca, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+menopause+or+early+menopause:+Long-term+health+consequences.">Premature menopause or early menopause: Long-term health consequences.</a> Maturitas. 65 (2), 161-166 (2010).</li><li>Jacobsen, B. K., Knutsen, S. F., Fraser, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+at+natural+menopause+and+total+mortality+and+mortality+from+ischemic+heart+disease:+The+Adventist+health+study.">Age at natural menopause and total mortality and mortality from ischemic heart disease: The Adventist health study.</a> J Clin Epidemiol. 52 (4), 303-307 (1999).</li><li>Kulaksizoglu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+Factors+for+Diabetes+Mellitus+in+Women+with+Primary+Ovarian+Insufficiency.">Risk Factors for Diabetes Mellitus in Women with Primary Ovarian Insufficiency.</a> Biol Trace Elem Res. 154 (3), 313-320 (2013).</li><li>Johnell, O., Kanis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+estimate+of+the+worldwide+prevalence+and+disability+associated+with+osteoporotic+fractures.">An estimate of the worldwide prevalence and disability associated with osteoporotic fractures.</a> Osteoporos Int. 17 (12), 1726-1733 (2006).</li><li>Rosario, E. R., Chang, L., Head, E. H., Stanczyk, F. Z., Pike, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+levels+of+sex+steroid+hormones+in+men+and+women+during+normal+aging+and+in+Alzheimer's+disease.">Brain levels of sex steroid hormones in men and women during normal aging and in Alzheimer's disease.</a> Neurobiol Aging. 32 (4), 604-613 (2011).</li><li>Cargill, S. L., Medrano, J. F., Anderson, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infertility+in+a+line+of+mice+with+the+high+growth+mutation+is+due+to+luteal+insufficiency+resulting+from+disruption+at+the+hypothalamic-pituitary+axis.">Infertility in a line of mice with the high growth mutation is due to luteal insufficiency resulting from disruption at the hypothalamic-pituitary axis.</a> Biol Reproduction. 61 (1), 283-287 (1999).</li><li>Mason, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+young+ovaries+restored+cardioprotective+influence+in+postreproductive-aged+mice.">Transplantation of young ovaries restored cardioprotective influence in postreproductive-aged mice.</a> Aging Cell. 10 (3), 448-456 (2011).</li><li>Mason, J. B., Terry, B. C., Merchant, S. S., Mason, H. M., Nazokkarmaher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+Ovarian+Function+Significantly+Influenced+Trabecular+and+Cortical+Bone+Volume,+Architecture+and+Density+in+Mice+at+Death.">Manipulation of Ovarian Function Significantly Influenced Trabecular and Cortical Bone Volume, Architecture and Density in Mice at Death.</a> Plos One. 10 (12), 15 (2015).</li><li>Peterson, R. L., Parkinson, K. C., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+Ovarian+Function+Significantly+Influenced+Sarcopenia+in+Postreproductive-Age+Mice.">Manipulation of Ovarian Function Significantly Influenced Sarcopenia in Postreproductive-Age Mice.</a> J. Transplant. , (2016).</li><li>Peterson, R. L., Parkinson, K. C., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+immune+and+renal+function+in+aged+females+by+re-establishment+of+active+ovarian+function.">Restoration of immune and renal function in aged females by re-establishment of active ovarian function.</a> Reprod. Fertil. Dev. , (2017).</li><li>Parkinson, K. C., Peterson, R. L., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+behavior+and+sensory+function+were+significantly+influenced+by+restoration+of+active+ovarian+function+in+postreproductive+mice.">Cognitive behavior and sensory function were significantly influenced by restoration of active ovarian function in postreproductive mice.</a> Exp. Geront. 92, 28-33 (2017).</li><li>Cargill, S. L., Carey, J. R., Muller, H. G., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+of+ovary+determines+remaining+life+expectancy+in+old+ovariectomized+mice.">Age of ovary determines remaining life expectancy in old ovariectomized mice.</a> Aging Cell. 2 (3), 185-190 (2003).</li><li>Brooks, H. L., Pollow, D. P., Hoyer, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VCD+Mouse+Model+of+Menopause+and+Perimenopause+for+the+Study+of+Sex+Differences+in+Cardiovascular+Disease+and+the+Metabolic+Syndrome.">The VCD Mouse Model of Menopause and Perimenopause for the Study of Sex Differences in Cardiovascular Disease and the Metabolic Syndrome.</a> Physiology. 31 (4), 250-257 (2016).</li><li>Rivera, Z., Christian, P. J., Marion, S. L., Brooks, H. L., Hoyer, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroidogenic+Capacity+of+Residual+Ovarian+Tissue+in+4-Vinylcyclohexene+Diepoxide-Treated+Mice.">Steroidogenic Capacity of Residual Ovarian Tissue in 4-Vinylcyclohexene Diepoxide-Treated Mice.</a> Biol. Reprod. 80 (2), 328-336 (2009).</li><li>Yuan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+inbred+strains+of+mice:+study+design+and+interim+report+on+median+lifespans+and+circulating+IGF1+levels.">Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels.</a> Aging Cell. 8 (3), 277-287 (2009).</li><li>Mason, J. B., Cargill, S. L., Anderson, G. B., Carey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Young+Ovaries+to+Old+Mice+Increased+Life+Span+in+Transplant+Recipients.">Transplantation of Young Ovaries to Old Mice Increased Life Span in Transplant Recipients.</a> J. Gerontol. A Biol. Sci. Med. Sci. 64 (12), 1207-1211 (2009).</li><li>Harrison, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acarbose,+17-alpha-estradiol,+and+nordihydroguaiaretic+acid+extend+mouse+lifespan+preferentially+in+males.">Acarbose, 17-alpha-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males.</a> Aging Cell. 13 (2), 273-282 (2013).</li><li>Manay, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17&#945;-Estradiol+Attenuates+Neuron+Loss+in+Ovariectomized+Dtg+A&#946;PP/PS1+Mice.">17&#945;-Estradiol Attenuates Neuron Loss in Ovariectomized Dtg A&#946;PP/PS1 Mice.</a> J. Alzheimers Dis. 23 (4), 629-639 (2011).</li><li>Parker, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+conservation+at+the+time+of+hysterectomy+and+long-term+health+outcomes+in+the+nurses'+health+study.">Ovarian conservation at the time of hysterectomy and long-term health outcomes in the nurses' health study.</a> Obstet Gynecol. 113, 1027-1037 (2009).</li></ol>

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&#34; 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&#34; 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+&#8482; Cordless Detachable Blade Clipper and a CeramicEdge&#174; #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 ...

The overall goal of these procedures is to separate the influence of reproductive aging from chronological aging in females. This method can help answer key questions in the biomedical field such as how do ovarian signals from young ovaries preserve healthy young females, and restore health in menopausal females? The main advantage of this technique is that we can transfer components of ovarian signaling from a young healthy animal to an older animal that has lost its young signaling.The implications of this technique extend toward the therapy of aging associated disease. As many of these diseases can be avoided in old mice through the restoration of young ovarian signaling. Though this method can provide insight into menopause associated increases in disease it can also be applied to other disease such as those associated with primary or secondary ovarian insufficiency.Before beginning the procedure confirm the appropriate level of sedation by toe pinch. And apply ointment to the animal's eyes. Place the mouse in the supine position on a stack of heated paper towels and starting just below the ribs and moving distally use number 40 blade hair clippers to remove a two to three centimeter square of hair, a few millimeters lateral to the midline on each side of the animal with a one to two centimeter strip of hair covering the midline.After administering analgesia transfer the animal onto a heated sterile surgical field in the supine position, and use a two by two centimeter piece of 70%ethanol soaked gauze followed by a Betadine to disinfect the surgical area. Next make a one to 1.5 centimeter paralumbar incision in one of the surgical areas and use scissors to blunt dissect the skin from the underlying fascia. Then make another incision through the fascia and blunt dissect laterally under the fascia as superficially as possible until the abdominal cavity is reached.Place a two by two centimeter gauze surgical drape over the incision and wet the gauze with sterile saline. Using small forceps locate the adipose tissue that surrounds the ovary and gently extract the fat pad and ovary onto the drape. The ovarian fat pad will appear whiter than the surrounding adipose tissue.Place a 1.5 inch bulldog clamp onto the fat pad taking care not to obstruct the ovarian bursa to hold the tract in place during the procedure. Use a dissecting microscope to identify the ovarian bursa. Then grasp the bursa with two watchmaker forceps and incise the bursa on the opposite side from the ovarian hilum to expose the ovary.Before deciding where to open the bursa try to visualize a pocket in the bursa where you will place the new ovary prior to suturing. Only open the bursa as far as necessary to remove the endogenous ovary. Gently remove the ovary from the bursa and clamp the ovarian hilum with watchmaker forceps to excise the ovary and to prevent bleeding.Then place the donor ovary in a watch glass containing four degrees celsius sterile saline and place a loose cover over the glass. After ovariectomizing the recipient mouse as just demonstrated place a small piece of hemostatic foam pad inside the empty bursa of the recipient animal to control bleeding and place a small piece of saline soaked gauze over the exposed surgical site. Load a castroviejo needle holder with a tapered needle threaded with a nine O or 10 O filament suture and glass bead for embedding.Using a second pair of watchmaker forceps simultaneously remove the gauze and foam pad from the recipient bursa. Then pick the glass bead with a Bishop-Harmon forceps and drop the bead onto the fat pad next to the bursa to allow the bead to be rolled into the bursa. When the bead is finally in place close the bursa with one to two Halsted sutures and lightly irrigate the site with sterile saline.After removing the fat pad clamp return the ovary and fat pad to the abdomen. Stay sutures may need to be placed to position the ovary for the subsequence placement of sutures for closing the bursa. Then use or vicryl sutures to close the abdominal wall and nine millimeter wound clips to close the skin.Finally the animal in a heated recovery cage shielded from light with monitoring until full recovery. Post-reproductive female mice that receive new ovaries live significantly longer than mice that undergo a sham surgery. No difference is detected between ovaries transplanted at the 11 or 17 months of age.However the depletion of germ cells prior to transplantation doubles the extension of the lifespan. The mean lifespan of CDAG female control mice is 644 days. Mice that undergo sham surgery at 11 months lived to an average age of 728 days.Acyclic intact mice live an average of 810 days when transplanted at 11 months. And 798 days when transplanted at 17 months. When the young ovaries are depleted of germ cells prior to transplantation the mean life span is extended to 880 days, twice the lifespan extension seen in mice that receive young germ cell containing ovaries.VCD treatment depletes primodial and primary follicles. The remaining secondary and antral follicles will then cycle and not be replaced, depleting the ovary of all germ cells. For example in this representative experiment ovaries collected from mice analyze 37 days after the initiation of VCD treatment demonstrate significantly depleted primordial and primary ovarian follicles compared to ovaries in control animals.After watching this video you should have a good understanding of how to manipulate ovarian function to facilitate separation of reproductive aging from chronological aging and be able to identify the separate influence of each on longevity and health span.

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

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

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...