STUDIES OF AUTOGRAFT OF CANINE OVARIES TO VARIOUS BODY
SITES
犬卵巣の自家異所移植に関する研究
寺園 司
2011
STUDIES OFAUTOGRAFT OF CANINE OVARIES TO VARIOUS BODY
SITES
AThesis
Submitted by
TSUKASA TERAZONO
Flor partial fulfillment of requirements fbr the degree of
Doctor of Philosophy
The United Graduate School of Vbterinary Sciences
Yamaguchi UniversitX Yamaguchi， Japan
2011
TABLE OF CONTENTS
ABSTRACT．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．1
GENERAL INTRODUCTION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．4
CHAPTER
Chapter 1：ASSESMENT OF CANINE OVARIES AUTOGRAFTED
TO VARIOUS BODY SITES．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．8
Abstract．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．8
1ntroduction．．．．．．．．．．．．．．．．．．．．．．．．．．＿．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．9
Materials and Methods．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．12
Results．．．．．．．．．．．．．．．．．．．．．．．．．．．＿．．．．．．．．．．．．．．．．．．．．．，．．．．．．．．．．．．．．．．．．17
Discussion．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．18
Chapter 2：FOLHCLE IN THE CANINE OVARY AFTER
AUTOGRAFTING TO A PERIPHERAL SITE．．．．．．．．．．．．．．．．．23
Abstract＿．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．23
1ntroduction．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．24
Materials and Methods．．．．．．．．．．．．．．．．．．．．．．．．．．＿．．．．．＿．．．．．．．．．．．25
Results．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．30
Discussion．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．32
OVERAH、 DISCUSSION AND CONCLUSION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．34
ACKNOWLEDGEMENT．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．39
TABLE AND FIGURES．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．40
REFIERENCES．．．．．．．．．．．．．．．．．．．．．．．．＿．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．47
ABSTRACT
Ovarian grafting may provide important insights into fbllicular development and
oocyte maturation． Several reports have described the heterotopic transplantation of
ovarian tissue but have not clearly identified the site that yields the best results afしer
transplantation． To our㎞owledge， the transplantation of canine ovadan tissue under
the skin of a bitch has never been reported． We investigated the in且uence of the gra負
site on the survival of canine fbllicles and oocytes and ex㎜ined fbllicular development
on the surface of canine ovarian tissue af㌃er autografting under muscle fascia and
meiotic resumption of fbllicle−derived oocytes afしer the maturation culture period．
The first series of experiments was conducted to investigate the influence of the
graft site on the survival of canine fbllicles and oocytes a負er autografting． Hemi−ovaries
were gra丘ed to 310cations（quadriceps fbmoris muscle fascia；kidney capsule；and
gastrosplenic ligament， the portion of dorsal mesentery between the spleen and the
stomach）and maintained fbr 28−31 days， after which they were recovered fヒom the
animals under anesthesia． The autografted hemi−ovaries were bisected：1／40f the
bisected portion of the ovary was used fbr histological assessment and the other， fbr
evaluating oocyte viability． The remaining portions of the丘esh hemi−ovaries were used
as controls to assess the viability of fbllicles and oocytes㎞the non−transplanted ovaries．
Most fbllicles in the histological sections of the gra丘s were classified as primordial or
primary． Antral fbllicles were not observed in the grafts， regardless of the graft site． The
percentages of viable fbllicles f士om the control ovaries and those grafted to the kidney
capsule， quadriceps fbmoris muscle fascia， and gastrosplenic ligament were l 7．4％，
22．9％，18．3％，and 32．4％， respectively． Twelve oocytes were recovered fセom the 15
1
hemi−ovaries grafted in 5 bitches， of which 5（41．7％）oocytes ffom autografts to the
quadriceps fbmoris muscle fascia and the kidney capsule were cultured fbr the
assessment of meiotic competence． Three oocytes were viable but remained in the
ge㎜inal vesicle stage a丘er a 72−h period of maturation culture． Results indicate that the
near−surface quadriceps fbmoris muscle fascia and kidney capsule might be usefUl
grafting sites， but fbllicle s㎜ival and meiotic competence of oocytes in the grafts must
be improved．
The second series of experiments evaluated the fbllicular development on the
surface of canine ovarian tissue after transplanting under the fascia near the surface of
the thoracol㎜b訂muscle and meiotic res㎜ption of鉛llicle−derived oocytes a食er the
maturation culture period． After ovarian excision fヒom a bitch， each ovary was cut
approximately in half The hemi−ovaries were transplanted into the original bitch at 3
diffヒrent body sites（under the fascia of the quadriceps fbmoris muscle， in the
thoracol㎜b訂muscles， and in the deltoid muscle of the scapul訂region）． All autogra且s
were recovered fヒom the bitch at 35 days afヒer transplantation． A visible antral fbllicle
was observed on the surface of the ovary grafヒed under the thoracolumbar fhscia．
Histological examination revealed viable fbllicles at diffbrent stages of development，
regardless of gra食site． Most granulosa cells in the fbllicles at dif驚rent stages of
development expressed proli色rating cell nuclear antigen（PCNA）． Three oocytes were
collected丘om an ovaワgra丘ed㎜der the血scia of the thoracol㎜bar muscle；loocyte
reached metaphase I after the maturation culture period． This report is the first of its
㎞nd to demonstrate fbllicular development and meiotic resumption of oocytes
recovered ffom autografted canine ovarian tissues．
2
Our results indicate that the peripheral魚scia and intr㎜uscular sites may be
suitable fbr grafting to monitor ovarian fUnction and recover oocytes fヒom the grafts．
The advantages of grafting to the pedpheral sites include grafting under local anesthesia，
easy monitoring by ultrasonography， and easy access fbr graft or oocyte recovery． The
use of both grafting and cryopreservation may prolong the lifヒspan of the fbmale
reproductive system．
3
GENERAL INTRODUCTION
Because of the particular physiology of the reproductive system in bitches， the
development of reproduction tec㎞010gies fbr dogs has lagged behind that of other
species． It is㎞own that bitches have a unique reproductive biology． For instance， the
bitch is monoestrous， with a 2−month luteal phase and a prolonged but variable
non−seasonal anestrus of 3−10 months at the end of both pregnant and non−pregnant
cycles． Canine oocytes at ovulation are at the ge㎜inal vesicle stage and require at least
48hto complete meiosis（Holst and Phemister，1971；Tsutsui，1989）， a period
considerably longer than the l 8 h and 24 h required fbr murine and bovine oocytes，
respectively（Randallθ’α1．，1990；Arlottoθ∫α1．，1996）． Befbre ovulation， basal levels of
se㎜progesterone begin to rise as luteinizing ho㎜one（LH）levels dse．0職lation
occurs approximately 2−3 days姐er the surge in LH（Wildtθ’01．，1979）． Infbmlation is
available regarding the timing of owlation relative to endocrine gland f㎞ction but is
limited regarding fbllicular growth during anestrus and proestrus． Although the
development of canine ovarian fbllicles can be observed by real−time ultrasonography
（Boydθ’α1．，1993；Englandθ’01．，1993）， no es槍blished method to accurately dete㎜ine
or predict ovulation is currently available． Moreover， imaging is tec㎞ically dif行cult
because of the location and small size of the ovaries． The meiotic competence of
oocytes collected fヒom ovaries of bitches at estnls has been demonstrated to be
significantly higher than that of oocyte s during the anestrus and diestrus stages
（Yamadaαα1．，1993；Hewitt and EngIand，1997；Otoiθ’α1．，2001）；however， the stages
of ovaries obtained by ovariohysterectomy in veterinary clinics are mainly anestrus or
diestrus， in which fbllicles are not visible on the surface of the ovary． An effbctive
4
procedure fbr in vitro maturation（IVM）of oocytes fヒom the anestrous and diestrous
stages is， therefbre， needed fbr ef丘cient in vitro embryo production．
For m㎝y species of ma㎜als， including dogs，飴llicul訂dyna血cs such as the
interval required fbr fbllicle growth and the hormonal re sponsiveness of ovarian
fbllicles remain unclear． In vitro study of fbllicular dyn㎜ics of primordial and primary
fbllicular stages is limited because in vitro culture systems fbr these fbllicles are lacking，
both in domestic animals and in humans． Several studies suggest that fbllicles in the
ovarian tissue can survive ovarian grafting， and they grow fヒom early to advanced
stages of development（Oktayαα1．，1998；Gosdenε’α1．，1994；Gunasenaθ’α1．，1998；
Weissmanθ’α1．，1999）；therefbre， ovarian gra丘ing can provide important insights into
fbllicular development and oocyte maturation． Several gra食ing tec㎞iques have been
reported， dif飴ring only in the location of the autografts， such as the bursal cavity， the
kidney capsule， and subcuta血eous sites． Subcutaneous ovarian grafting of驚rs several
advantages over intra−abdominal grafting（Oktay and Yih，2002）：the subcutaneous site
is more easily accessible， and monitoring fbllicle growth is relatively simple because
antral fbllicles in the graft can be studied by ultrasonography． In addition， subcutaneous
gra食ing allows fbr antral fbllicle development（Weissmanαα1．，1999）；however， the
kidney capsule and， to a lesser degree， the bursal cavity， became popular as
曾ansplantation sites because of the ass㎜ption血at the㎜ple blood supply in these
locations favored revascularization and sul）sequently， graft survival（Gosdenθ’α1．，
1994）．This is also the reason that recent research fbcuses on intramuscular grafting
together with incisions in the transplanted ovaries to improve blood supply（Eimaniθ’
α1．，2009）．Other authors believe that the transplantation site with respect to graf㌃
survival is not critical， as even cortical strips implanted subcutaneously achieve a rapid
neovascularization（Weissmanε∫α1．，1999）． Yangα01．（2006）reported that the
5
transplantation site can have a direct influence on the number and quality of oocytes
that can be harvested fヒom grafしed ovarian tissue．
Ovarian grafting to subcutaneous sites provides both a model to study canine
ovarian fUnction（Pris 8’α1．，2004）and ready access to the graft食）r oocyte collection
（Oktayα01．，2004；Parisε∫α1．，2004）． The low meiotic competence of canine oocytes
may be improved if oocytes can be collected fヒom the antral fbllicles of the ovarian
gra食s飴r in vitro飴質ilization（IVF）（Farstad，2000）． It is dif臼cult to prevent long−te㎜
r（jection of ovarian allografts in bitches that are monoestrous with a 2−month luteal
phase and have a prolonged non−seasonal anestrus of 3−10months；therefbre， autografts
appear to be the most promising approach fbr studying fbllicular development． An
important factor to consider in tissue survival and fbllicular development after
transplantation is the graft site（Soleimaniαα1．，2008）． Hypoxic conditions could be the
primary reason fbr initial fbllicle loss after transplantation（Aubard e’α1．，1999）；
therefbre， rapid revascularization after grafting is considered essential fbr maximum
飴11icle s㎜ival． Because of the ass㎜ption that adequate blood supply飴vors
revascularization and subsequent graft survival， the kidney capsule becam．e popular as a
transplantation site（Gosden 6’α1．，1994）． Although several reports have described the
transplantation of ovarian tissues to various heterotopic body locations（Gosdenε’01．，
1994；Weissmanαα1．，1999；Oktayθ’01．，2001；Schmidtαα1．，2005）， the site that
yields the best results after transplantation has not yet been clearly identified． In
addition， subcutaneous transplantation of canine ovarian tissue has never been studied
in bitches． Metcalfbθω1．（2001）transplanted canine ovarian tissue to the kidney
capsules of i㎜unode且cient mice， but鉤llicular development to the antral stage could
not be co面㎜ed．
6
In this study， we investigated whether the number of viable fbllicles after l month
of ovarian tissue transplantation is innuenced by the gra丘site． We also examined the
quality and meiotic competence of the oocytes recovered ffom the ovarian tissues
grafted to the quadriceps fbmoris muscle fhscia， kidney capsule， and gastrosplenic
lig㎜ent（the portion of dorsal mesentery between the spleen and the stomach）in dogs．
In the second study that involved ovarian grafting to subcutaneous sites， we
identified fbllicular development on the surface of canine ovarian tissue 35 days after
舳splanting under the血scia of the le丘thoracol㎜bar muscle． We con丘㎜ed meiotic
resumption of the fbllicle−derived oocyte afしer the maturation culture period．
7
Chapter l
ASSESMENT OF CANINE OVARIES AUTOGRAFTED TO VARUOUS
BODY SITES
ABSTRACT
The influence of graft site on the survival of canine R）llicles and oocytes after
autografting was investigated． Hemi−ovaries were autografted to three locations
（quadriceps fbmoris muscle fascia， kidney capsule， and gastrosplenic ligament）， and
grafted ovaries were recovered（under anesthesia）at 28−31 d after transplantation． The
grafted hemi−ovaries were bisected：one−quarter ovary was used fbr histological
assessment and another quarter fbr evaluation of oocyte viability． As controls， the
remaining ffesh hemi−ovaries were used to assess the viability of fbllicles and oocytes in
non−transplanted ovaries． Most fbllicles in the histological sections of the grafts were
classified as primordial or primary fbllicles． Antral fbllicles were not observed in the
grafts， irrespective of the graft site． The percentages of viable fbllicles in the sections
丘om control ovaries， and the ovaries grafted to the kidney capsule， the quadriceps
艶moris muscle fascia， and the gastrosplenic ligament were l 7．4％，22．9％，18．3， and
32．4％，respectively． A total of l 200cytes was recovered ffom the l 5 hemi−ovaries
grafted in five bitches， of which five（41．7％）oocytes f士om the ovaries grafted to the
quadriceps fbmoris muscle fascia and the kidney capsule， were cultured fbr assessment
of meiotic competence． Three oocytes were viable but remained in the ge㎜inal vesicle
stage afしer 72 h of maturation culture． The quadriceps fbmoris muscle fascia might be
usefUl fbr grafting like the kidney capsule， but improvement of fbllicle s㎜ival and
meiotic competence of oocytes in the grafts is necessary．
8
rNTRODUCTION
Several studies have reported that董）llicles in the ovarian tissue are able to survive
ovarian grafting， and that they grow f｝om early to advanced stages of fbllicular
development（Oktayε’α1．，1998；Go sdenθ’α1．，1994；Gunasenaθ’α1．，1998；Wissman
αα1．，1999）．Ovarian grafting should provide important insights into fbllicular
development and oocyte matmation． For many ma㎜ali㎝species， including dogs，
飴llicular dyn㎜ics such as the interval required fbr釦llicle gro舳and the ho㎜onal
responsiveness of ovarian fbllicles have not l）een elucidated． In bitches， because of the
pa而cular physiology of the色male gamete， development of reproduction tec㎞ologies
has lagged behind that of other species． Most reports describe the maturation rates of
canine oocytes cultured in vitro as extremely low（＜20％）（Farstad，2000）， which is a
m勾or obstacle to the in vitro production of canine embryos． In fact， most fbllicles in the
canine ovary remain below the ovarian surface， and they cannot be aspirated during the
anestrous and diestrous stages of the estrous cycle． ThereR）re， a possible reason負）r the
low maturation rate of oocytes is the use of oocytes with a small diameter（＜l l Oμm），
which were obtained ffom deeper corticaHbllicles（Otoiε’α1．，2000）． Ovarian grafts
mder the skin facilitate access to the gra丘fbr oocyte collection（Oktay and Yie，2002；
Oktayθ∫01．，2003）， and completion of the meiotic maturation of oocytes may be
increased if oocytes were collected丘om the antral fbllicles of the ovarian grafts fbr in
vitro飴貰ilization． Infb㎜ation about the timing of ovulation relative to endocrine gland
㎞ction is available， but there is limited infb㎜ation about fbllicular gro舳during
anestms and proestrus． Although it is possible to observe the development of canine
ovarian fbllicles by real−time ultrasonography（Boyd et al．，1993；England and Yeager，
1993），amethod to accurately dete㎜ine or predict ovulation has not been established．
9
Moreover， the location and small size of the ovahes make imaging tec㎞ically di伍cult．
Ovarian grafts under the skin facilitate monitoring of fbllicle growth（Oktayθ’α1．，2004）．
Ovarian grafting to the subcutaneous sites provides a model to study canine ovarian
fUnction（Parisαα1．，2004）；however， no studies have been perfb㎜ed in bitches on the
㎜der−skin transplantation of canine ovarian tissue． Allografts and xenografts can
survive inde且nitely in i㎜unodeHcient or i㎜unosuppressed hos伽imals（Pari sθ∫01．，
2004；Soleimaniθ’α1．，2008）． Canine pre−pubertal ovarian allografts transplanted into
血ebmsa of the recipient with i㎜mosuppressive therapy have resulted in pregn㎝cy
（Pulliumθ’α1．，2008）；however， this implies that i㎜unosuppressive therapy is
required fbr the survival of ovarian allografts． In bitches， who are monestrous with a
2−mo luteal phase and a prolonged non−seasonal anestnls of 3−10 mo， it is dif行cult to
prevent long−te㎜r匂ection of ovadan allogra負s． Therefbre， autogra丘s appear as the
most promising approach f∼）r studying fbllicular development． The graft site has been
considered an important factor in tissue survival and fbllicular development a負er
廿ansplantation（Soleimaniαα1．，2008）． Several ovarian gra食ing tec㎞iques have been
described；these basically diffbr only in the graft recipient site， which includes the
bursal cavity， kidney capsule， and subcutaneous sites． Hypoxic conditions might be the
main reason fbr initial fbllicle loss after transplantation（Aubardθ’α1．，1999）；therefbre，
rapid revascul肛ization a食er gra丘ing is considered necessaη飴r maxim㎜飴11icle
survival． The kidney capsule became popular as a transplantation site， because of the
ass㎜ption that adequate blood supply血vored revascularization and subsequent gra食
survival（Gosden α α1．， 1994）． To date， several reports have de scribed the
transplantation of ova1ian tissues to various heterotopic body locations（Gosdenαα1．，
1994；Weissmanθ’α1．，1999；Oktay et al．，2001；Sc㎞idt et al．，2005）， but have not
clearly identified the site that yields the best results afしer transplantation． In this study，
10
we investigated whether the gra食site innuences the n㎜ber of viable飴llicles a丘er l
mo of ovarian tissue transplantation， and examined the quality and meiotic competence
of the oocytes recovered fヒom the ovarian tissues grafted to the quadriceps fbmoris
muscle fascia， kidney capsule， and gastrosplenic ligament in dogs．
11
MATERIALS AND METHODS
1．Experimental animals
Beagle bitches（age，4−7 yr；mean weight，10．3土2．O kg）in a closed breeding
colony were used fbr this study． The dogs were housed individually in stainless steel
cages，角d a diet of stand肛d co㎜ercial dog鉛od once a day， and given water ad
libit㎜． All the animals involved in this study received humane care in compliance with
the Guide fbr the Care and Use of Laboratory Animals prepared by the Institute of
Laboratory Animal Resources， National Research Council． All procedures were
approved by the細imal Rese肛ch Co㎜i賃ee of Y㎜aguchi Universi取．
2．Transplantation procedure
The dogs were fasted fbr 12 h befbre the surgical procedure． At 30 min befbre
induction of anesthesia，0．2 mg／kg meloxic㎜（Nippon Boe㎞nger Ingelheim Co． Ltd．，
Hyogo， Japan）was admini stered subcutaneously fbr analgesia． General anesthe sia was
induced with an intravenous ir噸ection of O．2 mg／kg midazolam hydrochloride（Astellas
Pha㎜a Inc．， Tokyo， Japan）mixed with O．2 mg／kg buto1phanol ta直rate（Me麺i Seika Co．
Ltd．， Tokyo， Japan），鉛llowed by 4 mg／kg propo飴1（FゆPha㎜aceutical Co． Ltd．，
Toyama， Japan）． After endotracheal intubation， the dogs were mechanically ventilated
with isoflurane in pure oxygen and placed in dorsal recumbency． The carotid and
飴moral a貢ehes Imd the jugular and艶moral veins were㎜ilaterally catheterized fbr
monitoring the arterial and venous blood pressure． During the operation，5％glucose in
acetated Ringer’ssolution（Nikken Chemicals Co． Ltd．， Tokyo）was infUsed at a rate of
10mL／kg／h． Bilateral ovariectomy was per飴㎜ed using a ventral midline abdominal
approach starting at the umbilicus and extending caudally（Janssens and Janssens，1991）．
12
The ovarian pedicle was ligated using routine tec㎞iques and matedals． The uterine
artery and vein were then ligated and severed at the proper ligament（cranial tip of the
uterine hom）， and then， the ovary was removed． After excision， each ovary was bisected
using a scalpel blade（Fig．1−la）． The hemi−ovaries were stored in physiological saline
and maintained at 38°C． They were then transplanted to the animal of origin in three
locations within 20 min of ovary excision． As a control， the remaining hemi−ovary was
used to ex㎜ine ovarian morphology and the vial）ility of oocytes of fヒesh
non−transplanted ovaries．
（1）Kidney capsule
The left kidney was exteriorized t㎞ough ventral median celiotomy during
ovariectomy． A hemi−ovary was inserted under the capsule of the kidney through a
small incision（about 2 cm）made with a scalpel blade and iris scissors． After insertion
of the ova！y， the incision was closed with mono且lament nylon（De㎜alon 5−0；Davis＆
Geck Inc．， NJ， USA）．
（2）Gastrosplenic ligament
The gastrosplenic ligament near the splenic artery was exteriorized through ventral
median celiotomy． A hemi−ovary was placed in an invagination in the gastrosplenic
ligament， which was then closed with monofilament nylon in a purse−string suture
pattem
（3）Quadriceps femoris muscle fascia
13
Ahemi−ovary was inse貫ed㎜der the fascia of the le食quadriceps色moris muscle
through a small incision（about 2 cm）made with a scalpel blade and iris scissors． After
insertion of the ovary， the incision was closed with monofilament nylon．
Finally， the abdominal wall and the skin incisions were closed． All procedures were
per鉛㎜ed under aseptic conditions． All dogs recovered丘om anesthesia uneventfUlly
and were allowed fbod and water 6 h after recovery． All grafted ovaries were recovered
丘om animals at 28−31 d after transplantation mder anesthesia， as previously described．
After collection， each hemi−ovary was bisected using a scalpel blade， and one−quarter
ovary was used fbr histological assessment and the other fbr evaluation of oocyte
viability．
3．Histological assessment
The quarter ovaries ofthe recovered grafts and control tissues were fixed in 10％
R）mlaldehyde and manually processed fbr paraffin embedding． Histomoq）hological
ex㎜ination was conducted a丘er sectioning 4一μm slices． Then， two serial sections with
maximal area丘om each graft and control tissue were selected fbr hematoxylinreosin
（HE）（Fig．1−2a）and prolifbrating cell nuclear antigen（PCNA）staining（Fig．1−2b）． For
PCNA i㎜㎜ohistochemist琢， according to止e m舳c紺er’s instmctions of the
Histofine SAB−PO kit（Nichirei CoΦ．， Tokyo， Japan）， tissue sections（4μm）were
dewaxed and rehydrated using ascending concentrations of alcohol befbre undergoing
the㎜al antigen retrieval in citrate buf色r（10 mM；pH 6．0）． Slides were incubated at
25°Cfbr 90 min with anti−PCNA mouse monoclonal antibody（38 mg／mL；PCNA
Clone PClO；Sigma， St． Louis， MO， USA）， which was diluted l：100 in PBS．
Biotinylated rabbit anti−mouse IgG， IgA， and IgM antibody（Nichirei Corp．）was
14
applied as a secondary antibody fbr 30 min befbre treatment with peroxidase−cor巾gated
streptavidin（Nichirei Corp．， Tokyo， Japan）． After treatment with a chromogenic
substrate（3−amino−9−ethylcarbazol）， the sections were co㎜terstained with hematoxylin．
Because PCNA is not expressed in the quiescent primordial fbllicles and marks the
initiation of fbllicular growth（Oktayε’01．，1995）， the fbllicles with granulosa cells
expre ssing PCNA in the section were considered viable． In both HE−and PCNA−stained
sections， fbllicles were classified as primordial（oocytes s㎜ounded by one layer of
nattened pre−granulosa cells）， primary（surrounded by one layer of cuboidal granulosa
cells）， secondary（with two or more layers of granulosa cells without an antrum）， or
antral（with multiple layers of cuboidal granulosa cells and an antral cavity）（Oktayθ’
α1．，1995）．In PCNA−stained sections， the total n㎜ber of飾llicles and the n㎜ber of
fbllicles with granulosa cells expressing PCNA were comted． The percentage of viable
鉛llicles was calculated by dividing the n㎜ber of PCNA−positive鉤llicles by the total
number of fbllicles． To prevent counting errors， slides were assessed by two
independent individual s， and the re sults were compared．
4．Oocyte viabi屠ity
The quarter ovaries of recovered grafts and control ovaries were placed in modified
phosphate−buf琵red saline（m−PBS；Nippon Zenyaku Kogyo Co． Ltd．， Fukushima，
Japan）supplemented with 50μg／mL gentamicin（Sigma）at 38°C． They were then
repeatedly sliced to release oocytes． All oocytes were collected and suspended in culture
medi㎜（TCM 199 with E肛lels salts）， bu価red wi血25 mM HEPES bu舳r（Invitrogen
Corp．， Carlsbad， CA， USA）， and supplemented with l O％（v／v）fbtal bovine semm
（FBS；Invitrogen Corp．）and 50μg／mL gentamicin． The oocytes were microscopically
ex㎜ined fbr mo仙ological quali妙and categorized using the飴llowing method by
15
Hewittθ’α1．（1998）：Grade A oocytes were darkly pigmented and surrounded by one or
more layers of c㎜ulus cells． Grade B ooc外es were lightly pigmented with incomplete
layers of c㎜ulus cells． Grade C oocytes were pale and iπegularly shaped， with no
c㎜ulus cells． Only grade A and B oocytes were selected and trans色πed into lOO一μL
drops of cul伽medi㎜， and covered with w㎜即笛n oil（3。5 mL， Sigma）in a
polys取rene cul加re dish（35×10㎜；Falcon Becton Dickinson Labw肛e， NJ， USA）．
Subsequently， the oocytes were cultured鉤r 72 h at 38．5°C in a h㎜idi且ed a㎞osphere
of 5％CO2 in air．
After incubation fbr 72 h， the oocytes were denuded using small glass pipettes， and
且xed and pe㎜eabilized fbr 15min at room temperature in Dulbecco’sPBS（DPBS；
Invi甘ogen CoΦ．）supplemented with 3．7％（w／v）parafb㎜aldehyde and l％（v／v）Triton
X400 （Sigma）． Then， they were placed in PBS containing O．3％ （w／v）
polyvinylpyrrolidone fbr l 5 min at room temperature． The oocytes were transfbrred into
asmall drop comprising PBS supplemented with 90％（v／v）glycerol（Sigma）and 1．9
μMbisbenzimide（Hoechst 33342；Sigma）on a slide． Subsequently， the oocytes were
overlaid with a coverslip supported by fbur droplets of vaseline／paraffin and incubated
ovemight at 4°C． The oocytes were examined using且uorescence microscopy with a
355一㎜wavelength excitation且lter． They were classi負ed according to their chromatin
co面guration as“ge㎜inal vesicle，”“condensed chromatin，”“metaphase I，”or
‘‘
metaphase II．”Oocytes with diffUsely stained cytoplasmic characteristic of nonviable
cells， and those in which chromatin was㎜identi盒able or not visible， were classi且ed as
‘‘
degenerated．”
16
RESULTS
1．Histological assessment
One month after transplantation， all ovarian tissues were surrounded by a layer of
fibrous tissue and blood vessels， hTespective of the graft site（Fig．1。1b）． In both］E【E−
and PCNA−stained graft sections， most fbllicles were classified as primordial or primary
fbllicles， and antral fbllicles were not observed in the grafts， irrespective of the graft
location． The percentages of viable R）llicles in the sections fヒom the control ovaries
（total number of counted fbllicles，409 fbllicles）， and the ovaries grafted to the kidney
capsule（170 fbllicles）， the quadriceps fbmoris muscle fascia（224 fbllicles）， and the
gastrosplenic ligament（148 fbllicles）were 17．4％，22．9％，18．3％， and 32．4％，
respectively．
2．Ouality and viability of oocytes
Atotal of 1200cytes was recovered丘om the quarter ovaries of the 15 hemi−ovaries
grafted in five l）itches， of which five（41．7％）oocytes， which were recovered倉om the
ovaries grafted to the quadriceps fbmoris muscle fascia and the kidney capsule， were
categorized as grade A or B（Table l−1 and Fig．1−2c）． After in vitro maturation（IVM）
culture， three oocytes were stained by bisbenzimide， but remained at the GV stage（Fig．
1−2d）． No oocyte reached metaphase II（MII）． In the non−grafted controls，6400cytes
were recovered丘om the quarter ovaries of five hemi−ovaries， of which 33（51．6％）
oocytes were categorized as grade A or B． Only one out of 3300cytes reached MII a丘er
IVM．
17
DISCUSSION
In this study， canine hemi−ovaries were autografted to three locations to compare the
survival of the fbllicles and oocytes． After l mo of ovarian transplantation， growth of
lbllicles in the ovarian tissues could be observed， irrespective of the graft site． Moreover，
some viable oocytes were recovered fヒom the ovaries grafted to the quadriceps fbmoris
muscle fascia and to the kidney capsule， although the oocytes were㎜able to resume
meiosis after IVM culture．
The graR site has been proposed to influence tissue survival and fbllicle
development， as well as the quality of the oocytes obtained， after transplantation（Yang
θ’α1．，2006）．Clearyθ’01．（2003）reported increased fbllicle survival after grafting under
the kidney capsule compared to that observed after grafting to the subcutaneous sites in
xenografted ovarian tissues． Yangθ’α1．（2006）demonstrated that a significantly higher
number of oocytes were recovered fヒom the ovaries grafted under the kidney capsule
than fヒom the ovaries grafted to the subcutaneous sites． Post−transplantation hypoxic
ischemia has been suggested as the main cause of fbllicular loss in ovarian
transplantation （Dissen ε’α1．， 1994；Snow θ’α1．，2001）． Therefbre， rapid
revasculaization a丘er gra食ing is considered necessa琢飴r maxim㎜鉛llicle s㎜ival．
The duration of ischemia prior to revascularization of the transplanted ovarian tissue
depends on several factors such as the graft site， tissue size， and quantity of angiogenic
魚ctors（Parisα01．，2004；Bolsθ’α1．，2010）． The kidney has a rich blood supply and
high concentrations of angiogenic growth factors， which facilitate rapid blood vessel
ingrowth within 48 h of gra丘ing（Dissenθ’α1．，1994）． We transplanted the hemi−ovaries
to the kidney capsule and gastrosplenic ligament tissue． The gastrosplenic ligament is
the portion of dorsal mesentery between the spleen and the stomach． The vessels in the
18
ligament anastomose with the distal splenic vessels（Gurleyikθ’α1．，2000；Pauloθ’α1．，
2006）．Therefbre， an abundant blood supply， similar to that of the kidney， was predicted
when the hemi−ovaries were grafted to the tissue． In contrast， grafts in the less
vascularized recipient sites such as the subcutaneous sites might take longer to
revasculaize， thereby prolonging gra食ischemia㎝d reducing飴llicle s㎜ival（Imth㎜
ε’α1．，2000）．In this study， when the grafted ovaries were recovered， all ovarian tissues
were s㎜o㎜ded by a layer of丘brous tissue and blood vessels， irrespective of the gra丘
site． Moreover， the percentage of PCNA−positive fbllicles in the grafts under the
quadriceps fbmoris muscle fascia was similar to those in the fセesh control tissues and
other grafts． The back muscle has been demonstrated to provide superior supportive
neo−vascularization with specific blood supply to the graft site， thereby resulting in
increased s㎜ival of the ovarian grafしs than that at the kidney site（Soleimani e∫α1．，
2008）．Similarly， our results indicate that the quadriceps fbmoris muscle fascia may be
as suitable as the kidney capsule as a graft recipient site．
Ovarian grafting generates fbllicle development， and provides a model to study
ovarian fUnction． The kidney capsule has become popular as a transplantation site， but it
is dif行cult to access or monitor the tissues at this site． Peripheral graft sites such as the
蝕scia lata or the antebrachi㎜飴r ov肛ian tissue訂e more convenient and easily
accessible fbr transplantation surgery， monitoring of graft fUnction， and recovery of
oocytes ffom the grafts． In this study， the length of the hemi−ovaries transplanted under
the飴scia of the quadriceps色moris muscle could be monitored by ultraso㎜d， and the
mean di㎜eter of the grafted hemi−ovaries gradually decreased fヒom l．33 cm to O．93 cm
（decrease in diameter，0．4 cm）（data not sho㎜）． Approximately 18−32％of the fbllicles
co㎜ted in the gra丘sections was viable， but no antral fbllicles were observed in the
grafts． The number of oocytes recovered fヒom the grafted ovaries were much less than
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those recovered丘om the non−gra食ed ov訂ies， and no oocytes丘om the gra且s res㎜ed
meiosis． These results correlate with those of Metcalfθ’01．（2001）， who transplanted
c㎝ine ovari㎝tissue to the kidney capsules of i㎜unode且cient mice， but could not
conH㎜fbllicular development to the antral stage．
On the other hand， ca血e oocytes can res㎜e meiosis in vitro， but the e伍cacy of
the methods fbr IVM of oocytes is very low（Farstad，2000）． In this study， all oocytes
倉om the grafts arrested at the GV stage after IVM culture， and only 3％of control
oocytes could reach MII． The rate of maturation to MII of control oocytes was similar to
that in our previous study， in which the maturation rate of the canine oocytes fbm the
ovaries without visible fbllicles and pronounced luteal tissues was 2％（Otoiα01．，
2001）．In a previous study， ovarian autotransplantation with vascular anastomosis
yielded successfUl results with delivery of puppies（Paldiθω1．，1975）． Pulliumαα1．
（2008）demonstrated that bitches showed no㎜al estrous cycles and pregnancy a丘er
mating fbllowing homologous ovarian transplantation into the bursa of the recipient．
Moreover， Rimbachθ’α1．（1993）assessed the survival of canine transplanted ovaries
beyond 3 mo after grafting． In this study， all grafted ovaries were recovered fヒom
animals at 4 wk after transplantation， according to a previous study on
xenotransplantation of canine ovarian tissues（Suzukiθ’α1．，2008；Ish勾imaθ’α1．，2006）．
However， fblliculogenesis ffom the primordial to the preovulatory stage may take much
longer in larger animals than in rodents（Van den Broeckeαα孟，2001）． Therefbre， the
low n㎜ber of antral飴llicles㎝d meiotic competence of oocytes recovered丘om gra且s
may indicate that a coho質of fbllicles had beg㎜to grow but had had insu伍cient time
to expand or that a proportion of fbllicles of all size groups had died， leaving a small
n㎜ber of each取pe． Moreover， transplantation of the ov訂ian tissue to the heterotopic
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sites might a銑ct not only the n㎜ber but also the quali智and developmental potential
of oocytes（Yangα01．，2006）．
It has been suggested that血e timing of onset of㎝t㎜鉛mlation㎝d the progress
of fbllicle development are advanced relative to that in the ovaries in situ（Mattiskeθ’
α1．，2002）．In this study， when hemi−ovaries were autografted to each body site， no
fbllicles on the surface of each ovary were observed． One ovary had a corpus luteum，
and the concentration of progesterone in the donor’s semm， which was measured by an
enzyme−linked nuorescent assay using an autoanalyzer（SPOTCHEM VIDAS SV−5010；
Arkray， Kyoto， Japan）， was high （42．4 ng／mL）． However， the progesterone
concentration had decreased by the time of ovary recovery（0．28 ng／mL）． In other
bitches， no variations were observed in the concentrations of estradiol−17β （＜11．4
pg／mL）and progesterone（＜1．73 ng／mL）befbre and after grafting（data not sho㎜）．
These results indicate that luteal activity in all the grafts was quiescent at the time of
ovadectomy． The ovadan cycle of the bitch is mique among those of domestic animals，
in that the bitch is monestrous with a 2−mo luteal phase and a prolonged but variable
non−seasonal anestms of 3−10 mo after both pregnant and non−pregnant cycles． Unlike
most domestic species， the dog does not respond predic頓bly to ho㎜onal treatments
that induce ovulation in other species（lnabaθ∫01．，1998；Vanderlipθ’α1．，1987）．
Therefbre， it is not clear whether fbllicular development and ooc）雄e survival are related
to the endocrine status of donor or recipient at the time of transplantation． On the other
hand， the yo㎜g ovarian tissue contains more quiescent pdmordial and growing fbllicles
出an the adult ovarian tissue， and this tissue also飼㎜s more growing飴llicles than
adult donor gra食s． There飴re， it seems that antrum飴㎜ation and the progress of鉛llicle
development may be related to the age of the animal（Vom Saalαα1．，1994）．
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However， the influence of donor age on fbllicular development and oocyte survival
remains unclear fヒom our results， because only adult bitches with previous whelpi血g
experience（4 to 7 yr of age）were used fbr the experiment． Therefbre， fUrther analysis
of the role of endocrine status， age， and local factors in the control of fbllicle survival
befbre and after grafting is necessary to improve the development of fbllicles and
oocytes．
In conclusion， our results indicate that peripheral sites such as the fhscia lata may be
equivalent to the kidney capsule fbr transplantation． However， grafしs to the quadriceps
驚moris muscle fascia did not support early fbllicle and oocyte development． The
advantages of the peripheral recipient sites are that the grafts can l）e conveniently
inse並ed using local anesthetic， easily monitored by ultraso㎜d， Imd easily accessed fbr
grafl or oocyte recovery． Further investigation is necessary to improve the survival of
the fbllicles in the ovaries grafted to the peripheral sites．
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Chapter 2
FOLLICLE FORMATION IN THE CANINE OVARY AFTER
AUTOGRAFTING TO A PERIPHERAL SITE
ABSTRACT
This study reports about fbllicular development on the surface of canine ovarian
tissuea丘er autogra食ing under the飴scia of the thoracol㎜bar muscle and about meiotic
resumption of fbllicle−derived oocyte after maturation culture． After ovarian excision
仕om a bitch， each ovary of the pairs was cut approximately into half The hemi−ovaries
were transplanted into the bitch of origin at 3 diffbrent body sites（under the fascia of
the quadriceps fbmoris muscle and the thoracolumbar muscle， and in the deltoid muscle
in the scapular region）． All grafted ovaries were recovered fヒom the bitch at 35 days
post−transplantation． A visible antral fbllicle was observed on the surface of the ovary
gra丘ed under the thoracol㎜b肛飴scia． Histological ex㎜ination revealed viable
R）llicles at dif驚rent stages of development irrespective of gra丘site． Most granulosa
cells in the fbllicles at dif琵rent stages of development expressed prolifbrating cell
nuclear antigen（PCNA）． A total of 300cytes were collected丘om an ovary grafted
mder the飴scia of the thoraco1㎜b訂muscle， wherein an oocyte reached metaphase I
afしer maturation culture． This is the first report to demonstrate fbllicular development
and meiotic res㎜ption of oocytes recovered倉om autogra食ed canine ovarian tissues．
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INTRODUCTION
The reproductive biology of bitches has been㎞o㎜to be㎜ique． For instance，
ovulation occurs approximately 2−3 days a負er the surge in luteinizing ho㎜one（LH）
levels， and prior to o四lation， the se㎜progesterone levels begin to rise丘om basal
levels coincident with the LH peak（Wildtε’01．，1979）． Use血l in飴㎜ation about the
time of ovulation relative to the endocrine glands is available， but there is limited
infb㎜ation about鉛llicular gro舳through the anestrus and proestrus． Although it is
possible to observe the development of canine ovarian fbllicles by real−time
ultrasonography（Boyd e’α1．，1993；England and Yeager，1993）， a tec㎞ique to
accurately dete㎜ine or predict ovulation has not yet been established． Moreover， the
location and small size of ovades makes imaging tec㎞ically difHcult． On the other
hand， ovarian grafts under the skin allow easy monitoring of fbllicle growth and provide
ready access to the grafi fbr oocyte collection（Oktayε’α1．，2004）． Therefbre， ovadan
rafting to subcutaneous sites provides a model to study canine ovarian fUnction（Parisθ’
α1．，2004）．If oocytes can be collected f士om the antral fbllicles of the ovarian grafts fbr
in vitro fbrtlization， the low meiotic competence of canine oocytes may be improved
（Farstad，2000）． However， transplξmtation of canine ovarian tissue㎜der the skin has
never been investigated in the bitch． Metcal飴αα1．（2001）per飴㎜ed transplan愉tion of
canine ov訂ian tissue to the kidney capsules of i㎜unode且cient mice， but they could
not con丘㎜飴11icular development to the antral stage．
In the present study， we report fbllicular development on the surface of canine
ovarian tissue 35 days after autografting under the fascia of the left thoracolumbar
muscle． We co面㎜ed meiotic res㎜ption of血e bllicle−derived oocyte a丘er
maturatiOn CUItUre．
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MATERIALS AND METHODS
Bitch
Ahealthy beagle bitch（age，5years；weight，10．3 kg）used in this study was reared
in a 68 closed breeding colony and showed no㎜al estrous cycles（interestrous interval，
241days）and breeding（parity number，6）until the start of experiment． At the time of
ovariectomy， the concentrations of progesterone and estradiol−17βin the donor’s serum，
which were measured by an enzyme−linked f［uorescent assay using an autoanalyzer
（SPOTCHEM VIDAS SV−5010；Arkray， Kyoto， Japan）， were O．67 ng／mL and 9．6
pg／mL， respectively． The bitch was housed alone in a stainless steel cage（900 x 770 x
710㎜），and was given s伽d鍵d co㎜ercial dog飴od once a day，㎝d water， ad
libitum． She received humane care in compliance with the Guide fbr the Care and Use
of Laboratory Animals， prepared by the Institute of Laboratory Animal Resources，
National Research Council． All procedures were approved by the Animal Research
Co㎜i賃ee of Yamaguchi Universi取．
Transp書antation and recovery procedure
General anesthesia was induced using a mixture of isoflurane and oxygen；bilateral
ovariectomy was then perfb㎜ed according to the method descdbed by Janssens and
Janssens（1991）． Briefly，0．2 mg／kg meloxic㎜（Nippon Boehringer Ingelheim Co． Ltd．，
Hyogo， Japan）was administered subcutaneously fbr analgesia befbre induction of
anesthesia． General anesthesia was induced with an intravenous irj ection of O．2 mg／kg
midazolam hydrochloride（Astellas Pha㎜a Inc．， Tokyo， Japan）mixed with O．2 mg／kg
butorphanol tartrate（Me麺i Seika Co． Ltd．， Tokyo， Japan）， fbllowed by 4 mg／kg
propo飴1（F両i Ph㎜aceutical Co． Ltd．， Toy㎜a， Japan）． A丘er endotracheal intubation，
25
the dog was mechanically ventilated with isoflurane in pure oxygen and placed in dorsal
rec㎜bency． Bilateral ov雛iectomy was per鉛㎜ed using a ventral midline abdominal
approach starting at the umbilicus and extending caudally． The ovarian pedicle was
ligated using routine tec㎞iques and matehals． The ute血e artery and vein were then
ligated and severed at the proper ligament（cranial tip of the uterine hom）， and then， the
ovary was removed． After ovarian excision， each ovary of the pairs was removed fヒom
魚ttissues and then cut longitudinally into half by using a scalpel blade（Fig．2−1A）． The
hemi−ovaries were maintained in physiological saline at 38°C， and then transplanted in
the bitch of origin at 3 diffbrent 6 body sites within 20 min of ovary removal． As a
control， the remaining hemi−ovary was used to examine the fbllicular morphology and
oocyte viability of the丘esh non−transplanted ovary．
The hemi−ovaries were inserted under the f哀scia of the left quadriceps fbmoris
muscle and the left thoracolumbar muscle through a small hole（∼2 cm）made using a
scalpel blade and iris scissors． The third hemi−ovary was inserted into the deltoid
muscle in the left scapular region through a hole（∼5 cm）detached by an incision in the
vertical direction along the muscle fiber． Finally， the hole and skin incision were closed．
The longest diameters of the hemi−ovaries transplanted into the fascia and
intramuscular sites were measured every week until graft collection， with the Prosound
α7 ultrasound scanner（ALOKA Co．， Ltd．， Tokyo， Japan）equipped with a
6．0−13．0−MHz linear−array transducer．
All grafted ovaries were recovered fヒom the anesthetized bitch 35 days post−
transplantation， according to a previous study on xenotransplantation of canine ovarian
tissues（Ish勾imaθ’α1．，2006；Suzukiαα1．，2008）． After collection， each ovary was cut
approximately into half by using a scalpel blade． Histological assessment was carried
26
out on l half of each grafted hemi−ovary， and the other half was su切ected to oocyte
viability examination．
HistOlogical assessment
Half ofthe recovered graRs and control tissues were五xed in 10％鉛㎜aldehyde and
manually embedded in paraf丘n． Histomorphological examination was carried out afしer
sedal sectioning to 4一μm thic㎞ess， and 4 sections with maximal area丘om each gra負
and control tissue were selected and stained with hematoxylin−eosin（HE）and
proli鉛rating cell nucle冴㎝tigen（PCNA）． PCNA i㎜皿ohistochemical tec㎞que was
perfb㎜ed using a Histo且ne SAB−PO kit（Nichirei Corp．， Tokyo， Japan）， according to
the manufacturer’s instructions． Tissue sections were dewaxed and rehydrated using
ascending concentrations of alcohol befbre undergoing the㎜al antigen retdeval in
citrate buf驚r（10 mM；pH 6．0）． Slides were incubated at 25°C fbr 90 min with
anti−PCNA mouse monoclonal antibody（38μg／ml；PCNA Clone PC 10；Sigma， St．
Louis， MO， USA）， which was diluted l：100 in PB S． Biotinylated rabbit anti−mouse IgG，
IgA， and IgM antibody（Nichirei Corp．）was applied as a secondary antibody fbr 30 min
befbre treatment with peroxidase−corOugated streptavidin（Nichirei Corp．， Tokyo，
Japan）． After treatment with a chromogenic substrate（3−amino−9−ethylcarbazol）， the
sections were counterstained with hematoxylin． Mouse IgG（Dako De㎜飢k， Glostrup，
De㎜ark）as source of iπelevant prima理antibody was used as negative contro1．
Because PCNA is not expressed in the quiescent primordial fbllicles and marks the
ir盛tiation of fbllicular growth（Oktayθ’01．，1995）， the fbllicles with granulosa cells
expressing PCNA in the section were considered viable． In both HE−and PCNA−stained
sections， fbllicles were classified as primordial（oocyte surrounded by l layer of
nattened pre−granulosa cells）， primary（surromded by l layer of cuboidal granulosa
27
cells）， secondary（with 20r more layers of granulosa cells without an antrum）， or antral
（with multiple layers of cuboidal granulosa cells and an antral cavity）（Oktayθ’α1．，
1995）．In PCNA−stained sections， the total n㎜ber of鉤llicles㎝d the number of
fbUicles with granulosa cells expressing PCNA were counted． The percentage of viable
fbllicles was calculated by dividing the number of PCNA−positive fbllicles by the total
number of fbllicles．
Oocyte viability
Half of the recovered grafts and control ovaries were repeatedly sliced fbr oocyte
recovery， according to the method described by Otoi et al．（2002）with minor
modi且cations． All the oocytes were collected， and then， suspended in cul傭e medi㎜
（TCM 199 medium［Earlels salts］bu舳red with 25㎜ol HEPES bu制Invitrogen，
Carlsbad， CA， USA］supplemented with 10％（v／v）fbtal bovine serum ［FBS；
Invitrogen］and 50μg／ml gentamicin）． The oocytes were microscopically examined fbr
morphological quality， and categorized according to the fbllowing classification based
on the system described by Hewi賃et al．（1998）：Grade A oocytes were darkly
pigmented and s㎜ounded by l or more layers of cumulus cells；Grade B oocytes were
lightly pigmented with incomplete layers of c㎜ulus cells；Grade C oocytes were pale
㎝diπegulady shaped wi血out any c㎜ulus cells． Only Grade A and B oocytes were
selected and trans飴πed into 100一μL drops of culture medium covered with w㎜
para伍n oi1（3．5 mL；Sigma）in a polys取rene culture dish（35×10㎜；Falcon；Becton
Dickinson Labware， Franklin Lakes， NJ， U SA）． The oocytes were subsequently cultured
fbr 72 h at 38．5°C in a humidified atmosphere of 5％CO2 in air．
After 72−h incubation， the oocytes were denuded，且xed， and stained with 1．9μmol
bis−benzimide（Hoechst 33342；Sigma）on a slide（Otoiθ∫α1．，2002）． The oocytes were
28
examined using a nuorescence microscope with a 355一㎜wavelength excitatio面lter，
and classified according to chromatin configuration as‘‘ge㎜inal vesicle，”‘‘condensed
chromatin，”“metaphase I，”or‘‘metaphase II．”The oocytes with diffUsely stained
cytoplasmic characteristics of non−viable cells and those wherein the chromatin was
unidentifiable or not visible were considered degenerated．
29
RESULTS
Histological and ultrasonographic assessment of autografts
After autografting， all the ovarian tissues were surrounded by a thin layer of fibrous
tissue and blood vessels and they slightly diminished size irrespective of the grafting
body site（Fig．2−1C and D）． A visible antral fbllicle was noted on the surface of the
ovaW gra食ed㎜der the thoracol㎜bar飴scia（Fig 2−lB）． In HE s頓ined sections signs of
degeneration in blood vessels， such as nuclear py㎞osis， were observed in part in the
grafts． However， all grafts were well vascularized， had multiple growing fbllicles in the
cortex， and were surromded by a thin layer of且brous tissue irrespective of the gra丘ing
body site（Fig．2−2）． In both HE−and PCNA−stained graft sections（Fig 2−3）， most
R）llicles（78．2％−98．9％）in the control ovary（total number of counted fbllicles，87
fbllicles）， and the ovaries grafted to the quadriceps fbmoris muscle fascia （108
fbllicles）and the deltoid muscle（189食）llicles）were classified as primordial or primary
矧icles， whereas 76．5％of鉛llicles丘om an ovaW gra丘ed to the thoracol㎜bar魚scia
（34fbllicles）were classified as secondary fbllicles． The percentages of PCNA−positive
fbllicles in the sections ffom a control ovary， and the ovaries grafted to the quadriceps
艶moris muscle fascia， the deltoid muscle and the thoracolumbar fascia were 79．3％，
98．1％，100％，and 100％， respectively． The viable fbllicles were prominent in the cortex
of the grafted ovaries and the PCNA staining characteristics of preantral fbllicles were
similar to those of a control ovary．
Ultrasonograp㎞c assessment during gra食ing revealed that the longest di㎜eter of
the grafts under the fascia of the quadriceps fbmoris muscle gradually decreased f㌃om
l．46cm to 1．19 cm（decrease in di㎜eter，0．27 cm）． In the thoraco1㎜b肛飴scia and
30
deltoid muscle grafts， the decrease in the ovarian
di㎜eters were slightly lower（0．04
cm and O．11cm， respectively）．
Ouality and viability of oocytes from the autografts
Atotal of 300cytes that were classified as Grades A， B， and C， respectively， were
collected fヒom the ovary grafted under the fascia of the thoracolumbar muscle ffom
which a Grade A oocyte（Fig．2−lE）was recovered ffom the fbllicle fbmled on the
ovarian surface（Fig．2−1B）． After i n vitro maturation（IVM）culture， the oocyte derived
丘om the fbllicle reached metaphase I（Fig．2−lF）． Another oocyte classified as Grade B
remained in the gerlninal vesicle stage． A total of 500cytes were recovered丘om the
ovary grafted under the fascia of the quadriceps fbmoris muscle， all of which were
classified as Grade C． None of the oocytes was recovered丘om the deltoid muscle in the
scapular region． In the non。grafted control，800cytes were recovered丘om the fヒesh
ov町， only l of which was categorized as Grade A， and it remained in the ge㎜inal
vesicle stage a丘er IVM culture．
31
DISCUSSION
In the present study， we observed fbllicular development on the surface of canine
ovahan tissue gra丘ed under the thoracol㎜bar飴scia． Moreover， an oocyte丘om this
fbllicle reached metaphase I a丘er IVM culture． To our㎞owledge， although only one
bitch was examined in this study， this is the first report to demonstrate fbllicular
development and meiotic resumption of an oocyte recovered fヒom a visible antral
fbllicle on the surface of canine ovarian tissues that were graf㌃ed into the peripheral
sites．
Ovarian grafting with fbllicular development provides a model fbr studying ovarian
fUnction． Although the kidney capsule is a popular transplantation site， it is difficult to
access and monitor this site． Transplantation of the ovarian tissue to peripheral sites
allows the tissue to be placed at a more convenient and easily accessible site fbr
monitoring graft fαnction and oocyte recovery． In the present study， we observed
fbllicular development and some viable oocytes in the ovarian grafts． It has been
suggested that the grafヒing site influences tissue survival and fbllicular development as
well as the quality of the obtained oocytes after transplantation（Yangα01．，2006）． The
main cause of fbllicular graft loss in the process of ovarian transplantation has been
suggested to be post−transplantation hypoxic ischemia（Dissenε’α1．，1994；Snowθ’α1．，
2001）．Therefbre， rapid revascularization after grafting is considered essential fbr
maxim㎜飴llicular s㎜ival． The duration of ischemia prior to revasculari囮tion of the
transplanted ovarian tissue depends on several factors， including graft site， tissue size，
and the presence and amount of angiogenic factors（Parisθ’01．，2004；Bols 8’α1．，2010）．
The kidney has rich blood supply and high concentrations of angiogenic growth factors，
which allow rapid blood vessel in growth within 48 h of grafting（Dissenθ’α1．，1994）。
32
We transplanted hemi−ovaries into the less vascularized sites（the fascia and
intramuscular sites）that take longer to revascularize， thereby prolonging graft i schemia
㎝dreducing鉤llicula s㎜ival（Imth㎜θ’α1．，2000）． Moreover， all ovaies had some
corpora lutea at the time of ovariectomy， but the concentration of progesterone in the
donor’s se㎜was low（0．67 ng／皿）． No vadations were obse押ed in the concentrations
of progesterone（＜0．67 ng／mL）befbre and after grafting（data not shown）， indicating
that luteal activiW in all the gra丘s was quiescent． However， we co面㎜ed the presence
of viable fbllicles at diffbrent stages of development 35 days of grafting irrespective of
graft site． All the ovarian tissues were surrounded by a layer of fibrous tissue and blood
vessels． Although the graft diameters gradually decreased after the grafting， the size of
the ovarian tissue could be monitored by ultrasonography． It has been demonstrated that
peripheral graft sites such as the back muscle or the antebrachium are suitable fbr
ovarian allowgrafts（Oktayε’α1．，2001；Leeθ’α1．，2004；Solehnaniθ’01．，2008）．
Therefbre， our results indicate that the peripheral fascia and intramuscular sites may be
one of suitable grafting sites fbr ovarian fUnction monitoring and oocyte recovery fヒom
the gra負s． The advantages of gra丘ing to the peripheral sites include insertion mder
local anesthesia， easy monitoring by ultrasound， and easy access fbr graft or oocyte
recovery． Use of grafting and cryopreservation in combination may prolong the fbmale
reproductive lifbspan．
Confl ict of interest statement
None ofthe authors of this paper has a financial or personal relationship with people
or organizations that could inapPropriately influence or bias the content of this paper・
33
OVERALL DISCUSSION AND CONC五USION
In this study， the fbllowing results were achieved：（1）the percentage of
PCNA−positive fbllicles in the grafts under the quadriceps fbmoris muscle fascia was
similar to that in the control tissues and other grafts，（2）the size and revascularization of
the ovarian tissue could be easily monitored using ultrasonography afしer the grafting，（3）
fbllicular development was observed on the surface of canine ovarian tissue grafted
under the thoracolumbar fascia， and（4）an oocyte fヒom this fbllicle could reach
metaphase I a丘er IVM culture． Although only l bitch was ex㎜ined in this study， this is
血e五rst repo貫， to our㎞owledge， of飴llicular development and meiotic res㎜ption of
an oocyte recovered丘om a visible antral fbllicle on the surface of canine autograft
ovarian ti ssues that were transplanted to the peripheral sites． Taken together， our results
indicate that the peripheral fascia and intramuscular sites might be suitable areas fbr
monitoring ovarian fUnction and recovering oocyte s ffom the grafts．
Ovarian grafting results in fbllicle development（Coxε’α1．，2000）；therefbre， it
provides a model to study ovarian fUnction． In many wildli免species，㎞owledge about
fbllicular dynamics（e．g．， the interval requ廿ed飴r fbllicle gro舳and ho㎜onal
responsiveness of ovarian fbllicles）is uh㎞o㎜． This in鉛㎜ation is impo伽t if
assisted reproductive tec㎞ologies are to be employed to aid in the conservation of a
species． Ovadan biopsies taken while animals㎜dergo other surgehes or ovarian tissue
collected f±om a recently deceased fbmale can be used in graf㌃ing studies to provide key
㎞owledge about fbllicular dynamics of a species when access to that species fbr
research pulposes is limited． If the o切ective of ovarian gra丘ing is to restore long−te㎜
色rtility or collect large numbers of mature oocytes fヒom small pieces of ovarian grafts，
maximizing the n㎜ber of鉛llicles that suMve is paramount．
34
It has been proposed that graft site influences tissue survival and fbllicle
development， as well as the quality of the oocytes obtained， after transplantation（Yang
θ∫α1．，2006）．Clea【yθ’01．（2003）repo貢ed that fbllicle survival a食er gra丘ing㎜der the
kidney capsule was higher than that observed after gra負ing to the subcutaneous sites in
xenografted ovarian tissues． Yangα01．（2006）demonstrated that a significantly higher
number of oocytes were recovered丘om the ovaries grafted under the kidney capsule
than f｝om those grafted to the subcutaneous sites． Because hypoxic ischemia has been
suggested as the main cause of fbllicular loss afしer ovarian transplantation（Dissenθ’α1．，
1994；Snowθ’α1．，2001）， rapid post−grafting revascularization is considered to l）e
essential fbr maximizing fbllicle survival；the ischemic conditions cause fbllicles to
succ㎜b to apoptosis（Liuθ’α1．，2002）． Revascularization of ov訂i㎝gra丘s was
estimated to occur within 48 h of transplantation（Dissenθ’α1．，1994）， when the
m勾ority of growing fbllicles are lost and as much as 50％of the primordial fbllicle
population within an ovarian gra食do not survive（Liuθ’α1．2002）． This loss rate， in
cor蜘ction with cryopreservation， where about 9％of the fbllicle population is lost
（Liuε’α1．，2002）， and the initial small size of the graft， severely limits the lifbspan of
the graft． However， ovarian grafts can maintain reproductive fimction fbr many months
（Candyθ’α1．，2000）． Several factors such as the graft site， tissue size， and quantity of
angiogenic factors（Paris 8’α1．，2004；Bolsθ’α1．，2010）directly affbct the duration of
ischemia befbre revascularization of the autograft ovarian tissue． The kidney receives a
rich blood supply and has high concentrations of angiogenic growth factors， which
鉛cilitate rapid blood vessel ingrowth within 48 h of grafting（Dissenε’01．，1994）．
Promoting revascularization is the most obvious means fbr good graft establis㎞ent and
maxim㎜飴llicle s㎜ival． Vascular endothelial gro舳飴ctor（VEGF）and
trans飾㎜ing gro瓢h飴ctor（TGF−b）mRNA expression are upregulated during the
35
revascularization process of ovarian grafts（Dissenθ’α1．，1994）． This upregulation of
angiogenic stimulating factors may be mediated by pituita1y gonadotropins（Dissen e’
α1．，1994），implying why ovarian gra負s are more successfUl in bilaterally
ovariectomized recipients than in intact recipients（Coxθ’α1．，2000）． Treating the graft
recipient with a combination of FSH and LH 2 days befbre and 2 days after grafting
increased the number of fbllicles that survived within the ovarian graft（Imthum 8’01．，
2000）．Pres㎜ably， the e銑cts of F SH and田are mediated through VEGF；however，
VEGF administration to ovarian graft recipients does not appear to improve graft
establis㎞ent（Sc㎞orr e’α1．，2002）． Ischemic repe血sion irOu1y is also a problem with
ovarian grafting． Treatment of ovarian graft recipients with an antioxidant protects
fbllicles against such h埴ury（Nugent 8’α1．，1998）． Because of the inability of fbmale
ge㎜cells to undergo mitosis during postnatal development， optimization of
transplantation and cryopreservation protocols that minimize fbllicular depletion within
ovarian gra負s is essential if ovarian gra食ing is to be used as a long−te㎜solution鉤r
some cases of infbrtility．
In the first series of experiments， the hemi−ovaries were transplanted to the kidney
capsule and gas甘osplenic ligament tissue． The vessels in the lig㎜ent anastomosed with
the distal splenic vessels（Gurleyikθ’α1．，2000；Pauloαα1．，2006）；therefbre， an
abundant blood supply， similar to that of the kidney， was anticipated when the
hemi−ovaries were grafted to the tissue． In contrast， grafts in the less vascularized
recipient sites， such as the subcutaneous sites， might take longer to revascularize，
resulting in prolonged graft ischemia and reduced fbllicle survival（Inthumθ’α1．，2000）．
When we transplanted hemi−ovaries into less vascularized sites（i．e．， fascia and
intramuscular areas）， graft ischemia was prolonged and fbllicular survival was reduced
36
（Imthumθ’α1．，2000）；however， when the grafted ovaries were recovered， all ovarian
tissues were surro㎜ded by a layer of且brous tissue and blood vessels， regardless of the
graft site． In addition， the percentage of PCNA−positive fbllicles in the gra負s under the
quadriceps fbmoris muscle fascia was similar to that in the control tissues and other
grafts． These results indicate that both the near−sur￡ace quadriceps fbmoris muscle
飴scia and the kidney capsule might be usefUl fbr grafting．
Ovarian grafting with fbllicular development provides a model by which ovarian
hmction can 1）e studied． Although the kidney capsule is a popular transplantation site， it
is difficult to access and monitor． Transplantation of ovarian tissue to peripheral sites is
more convenient and more easily accessible fbr monitoring graft fUnction and oocyte
recovery． In both series of experiments， the hemi−ovaries transplanted under the fascia
of the quadriceps f6moris muscle and the other fhscia near the surface could be easily
monitored by ultrasonography． In the second experiment， an antral fbllicle with
fbllicular antrum fbmlation and blood vessels fヒom revascularization could also be
monitored by ultrasonography． In addition， in the行rst series of expehments， the n㎜ber
of oocytes recovered ffom the grafted ovaries was far lower than that recovered ffom
the non−gra丘ed ovades， and no oocytes丘om the gra丘s res㎜ed meiosis． These results
supported the report by Metcalfθ’α1．（2001）， wherein canine ovarian tissue was
伽spl㎝ted to the kidney capsules of i㎜mode且cient mice but could not con且㎝
fbllicular development to the antral stage． In contrast， in the second series of
experiments， an oocyte f卜om fbllicular development on the sur￡ace of canine ovarian
tissue gra丘ed under the thoracol㎜b曲scia reached me⑳hase I a丘er IVM culture．
Gene banks have been estal）lished worldwide to store reproductive tissues and cells
collected opportunistically丘om threatened wildlifb species；however， to be of potential
use， approphate methods are required飴r optim㎜utilization of such materials．
37
Ovarian transplantation is a valuable tool that may enable the generation of mature
oocytes ffom ovarian tissues salvaged fヒom endangered wildlifb species． Oocytes
hawested丘om gra丘s could then be used in IVF or intracytoplasmic spe㎜i両ection to
produce offヨpring． Of great advantage and in contrast to mature oocytes and embryos，
ovarian tissues can be collected ffom fbmales， irrespective of age and reproductive cycle，
even after recent death． These factors make ovarian transplantation an attractive assisted
reproductive tec㎞ique fbr use in the conservation endangered wildli飴species．
In recent ye飢s， non−h㎜an ovarian xenotransplan血tion studies have been extended
and the fbasibility of collecting oocytes fbom xenotransplants investigated， with oocytes
successfUlly recovered丘om ovarian transplants in wallaby， cow， mouse， and ma㎜oset
（Snowαα1．，2002；Parisθ’α1．，2004）． Fertilization and embryo cleavage have also been
achieved with oocytes harvested倉om wallaby， ma㎜oset， and mouse transplants to
角male recipients（Snowθ’α1．，2002；Parisθ’α1．，2004）． Imth㎜8’01．（2000）repo且ed
the制l potential of ovarian xenoαansplantation， co面㎜ing（a食er much speculation）
that live yo㎜g can be produced丘om xenogra丘一matured oocytes． In this cdtical study，
mouse ovarian tissue was xenotransplanted under the kidney capsule of bilaterally
ovariectomized nude mice recipients． At graft collection， oocytes were released ffom
the grafts， matured in vitro ovemight， and fbrtilized in vitro． Resulting 2−cell stage
embryos were transfbrred to fbster mothers that subsequently produced healthy live
young． This study suggested that oocytes that mature within ovarian grafts are nomlal
and capable of producing no㎜al，飴貫ile young．
In conclusion， the advantages of gra伍ng to peripheral sites include inse丘ion㎜der
local anesthesia， easy monitoring by ultrasonography， and easy access fbr graft or
oocyte recovery． By using a combination of grafting and cryopreservation， the lifbspan
of the fbmale reproductive system may be prolonged．
38
ACKNOWLEDGMENTS
The author gratefUlly thanks Profbssor Yasuho Taura， D．V．M．， Ph．D． of The United
Graduate School of Veterinary S cience， Yamaguchi University， fbr creating the pleasant
conditions necessary fbr the success ofthis work and fbr giving the various㎞owledge．
The author owes Associate Profbssor Kerji Tani， D．V．M．， Ph．D．， Associate
Pro飴ssor Mitsuhiro Takagi D．V．M．， Ph．D．， Pro飴ssor M㎜ekazu Nakaichi D．V．M．，
Ph．D．， Profbssor Makoto Inoue D．V．M．， Ph．D． and Profbssor Takeshige Otoi D．V．M．，
Ph．D． of The United Graduate School of Veterinary Science， Y㎜aguchi University， fbr
critical suggestion during the preparation of this dissertation．
The author has thanks very much to all of staff忌who have／had been concemed in
The United Graduate School of Veterinary Science， Yamaguchi University．
Some pa質s of this work were per鉛㎜ed at Terazono Animal Hospi愉l in Hiroshima．
The author would like to express thanks to all the colleagues of Terazono Animal
Hospital， Departlnent of Veterinary Reproduction and Surgery， Yamaguchi University，
Imd my丘iends． They gave me tec㎞ical and mental supports fbr this work．
Finally， the author is gratefUl to my wifb， Chitose Terazono，負）r taking care of my
sons， Daigo and Tak㎜i， and飴r suppo而ng the pleas㎝t conditions鉤r the success of
this work．
39
TABLE AND FIGURES
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Fig．2−1． Follicular development and oocyte recovery in an ovary grafted under the
飴scia of the thoracolumbar muscle．（A） Hemi−ovarian tissue prepared fbr
transplantation． The arrow indicates the corpus luteum．（B）ovarian tissue with
fbllicular development（arrow）35 days after grafting．（C）Grafしed ovarian tissue wlth
fbllicular development（arrow）and blood vessels（arrowhead）．（D）Cross section colour
Doppler sonogram of a 9−mm growing antral fbllicle with surrounding blood flow
（arrow）． Bars＝100μm．（F）The nuclear oocyte reached metaphase I after 72 h of
culture． The arrow indicates a metaphase chromosome．
44
し
＊
亀
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・、
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Fig．2−2． Histological section of ovarian tissue recovered 35 days after grafUng to the
quadriceps fbmoris muscle fascia． Hematoxylin and eosin（HE）stained tissue，
demonstrating a thin layer of fibrous tissue（L）surrounding the ovarian tissue， multiple
fbllicles in the cortex， and blood vessels（＊）． Ovarian cortex（C）and medulla（M）are
indicated in the image． Bar represents 500μm．
45
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C
L
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Fig．2−3． Histological appearance and expression of PCNA on ovarian images befbre and
a食er grafting． Hematoxylin and eosin（HE）stained tissues fセom the control ovary（A）
and the ovary graf㌃ed to the quadriceps fbmoris muscle fascia（B）， demonstrating
R）llicles in the early stages of growth． Insets represent magnified fbllicle cells． Ovarian
cortex（C）， medulla（M）， corpus luteum（CL）， and Hbrous tissue layer（L）are indicated in
Aand B images． Expression of prolifbrating cell nuclear antigen（PCNA）in the control
ovary（C）and the ovary grafied to the thoracolumbar muscle fascia（E）， and negative
control to C and E（D and F， respectively）， showing that most fbllicles at diffbrent stages
of development in grafted ovary displayed PCNA expression． Bars represent 500μm in
Aand B or 100μm in insets ofA， B and C−F．
46
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Ovarian Reserve in Women with BRCA Mutation

Live birth after autograft of ovarian tissue cryopreserved during

Addarii Lectures and Award 2014 - Policlinico S.Orsola

to cancer - Immunovaccine

Cancer - BWH Research Day

Brochure - Clinique OVO

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