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

Summary

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.

Abstract

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.

Introduction

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 close....

Protocol

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 t....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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