The 5th SRD-KSAR Joint Symposium“Reproductive Biology in the Next Decade”

September 9 (Wed), 2009, 12:15–16:30
Kinki University, School of Agriculture, Nara, Japan

Organizers:

Prof. Noboru MANABE (Chief Director of SRD)
Prof. Jong Taek YOON (President of KSAR)

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Program
Registration
11:30–
Opening Remarks
12:15–12:25

Prof. Akira IRITANI (Kinki University, Japan)

12:25–12:35

Prof. Kyung-Soon IM (Seoul National University, Republic of Korea)

Symposium Session
Chair persons: Noboru Manabe (The University of Tokyo, Japan)
Hoon-Taek LEE (Konkuk University, Republic of Korea)
12:40-13:05“Genetic clues of premature ovarian failure”
Youngsok CHOI (CHA University, Republic of Korea)

13:05-13:30“Regulation of growth hormone pulsatility in female goats: modulation by sex steroids
and involvement of neuropeptide Y”
Tomohiro YONEZAWA (Kitasato University, Japan)
Chair persons: Yoshihisa UENOYAMA (Nagoya University, Japan)
Jeong-Mook LIM (Seoul National University, Republic of Korea)

13:30–13:55“Factors affecting successful implantation and maintenance of pregnancy at thematernal-fetal interface in pigs”
Hakhyun KA (Yonsei University, Republic of Korea)

13:55–14:20“Neuroendocrine control of the pulsatile GnRH/LH secretion in ruminants”
Satoshi OHKURA (Nagoya University, Japan)--- Coffee brake ----

Chair persons: Takashi NAGAI (National Institute of Livestock and Grassland Science, Japan)
Youngsok CHOI (CHA University, Republic of Korea)
14:50–15:15“Developmental potential and reprograming efficiency of porcine embryos cloned withmesenchymal stem cells”
Gyu-Jin RHO (Gyeongsang National University, Republic of Korea)

15:15–15:40“Renewal of techniques related to embryo transfer in cattle – a novel superovulation
method using aluminum hydroxide gel”
Koji KIMURA (National Institute of Livestock and Grassland Science, Japan)

3Chair persons: Kiyoshi OKUDA (Okayama University)
Gyu-Jin RHO (Gyeongsang National University, Republic of Korea)
15:40–16:05“The ovary, as the source of stem cell”
Jeong-Mook LIM (Seoul National University, Republic of Korea)

16:05–16:30“A novel concept for the bovine luteolytic cascade with a focus on luteal blood flow andvasoactive factors”
Komei SHIRASUNA (Obihiro University, Japan)

Closing Remark
16:30–

Prof. Yukio TSUNODA (Kinki University, Japan)

4Genetic clues of premature ovarian failure
Youngsok CHOI*
Fertility Center of CHA Gangnam Medical Center, CHA University, Gangnam-gu, Seoul, Republic of Korea*E-mail address; youngsokchoi@cha.ac.kr

Premature ovarian failure (POF) is defined as a primary ovarian dysfunction characterized bydepletion of ovarian follicles before menopause in women. POF has an estimated prevalence of one
in 10,000 by age 20, one in 1000 by age 30, and one in 100 by age 40. POF is able to be heritable in
up to 30% of patients. POF results from both non-genetic and genetic problem. Non-genetic causes
include viral infection, gonadotoxic chemotherapy, radiation treatment, and autoimmune disorder.
However, in most case POF is idiopathic and probably genetic. Two chromosome abnormalities
such as Turner and fragile X-syndrome are known to be associated with POF. In addition
single-gene-mutation related to POF include Fragile X mental retardation 1 (FMR1), forkhead box
L2 (FOXL2), POF1B, inhibin alpha (INHA), growth differentiation factor 9 (GDF9), bonemorphogenetic protein (BMP15) and follicle stimulating hormone receptor (FSHR). However, thegenetic mechanism of POF is poorly understood. Recently Rajcovic laboratory added two
oocyte-specific transcriptional factors, newborn homeobox gene (NOBOX) and factor in germlinealpha (FIGLA), as candidate gene for premature ovarian failure in human. Mouse Nobox is
exclusively expressed in oocytes and encode a homeobox transcriptional regulator. Disruption ofmouse Nobox gene causes nonsyndromic ovarian failure in female mice, whereas males areunaffected. Nobox regulates several oocyte and germ cell-specific genes, Gdf9 and Pou5f1 (Knownas Oct4), through binding specific elements (TAATTG) on their promoters. In human, NOBOX is
preferentially expressed in the oocyte from primordial follicle through metaphase II oocytes. A
missense mutation of NOBOX, p.Arg355His, found in one of POF patient can disrupt NOBOX
binding affinity to the element (TAATTG). FIGLA, a basic helix-loop-helix (bHLH) transcriptional
factor, is expressed in the embryonic gonad. Figla regulates expression of zona pellucida genes aswell as that of other oocyte-specific genes. Like other bHLH transcriptional factors, Figla binds to a
specific element, E-box (CANNTG). Figla forms heterodimer with transcription factor 3 (TCF3) toregulate the zona pellucida gene promoters. Figla deficiency loses oocytes rapidly after birth,whereas male gonads are unaffected. A deletion c.419-421 delACA (p.140 delN) in human POF
patient was found. The deletion disrupted FIGLA binding to TCF3. These finding shows that
mutations in oocyte or germ cell-specific genes in women contribute to pathology of POF throughdiverse pathways. The addition of two transcriptional regulators as contributors to the etiology of
POF might be providing better understanding POF as well as genetic clues of POF.

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Regulation of growth hormone pulsatility in female goats: modulation
by sex steroids and involvement of neuropeptide Y
Tomohiro YONEZAWA1*, Kazutaka MOGI2, Jun You LI3 and Masugi
NISHIHARA4
1

Laboratory of Veterinary Physiology, Kitasato University, Towada, Japan

2

Companion Animal Research, Azabu University, Sagamihara, Japan

3

Animal Resource Science Center and 4Department of Veterinary Physiology, The University of Tokyo,

Tokyo, Japan*E-mail: yonezawa@vmas.kitasato-u.ac.jp

Growth hormone (GH) is secreted in a pulsatile manner, the pattern of which plays animportant role in the regulation of growth and metabolism. The Shiba goat, a Japanese miniature
goat, is a suitable experimental model for the analysis of GH pulsatility, because they have strictly
regular GH pulses. In female Shiba goats, the GH pulse frequency and amplitude in the follicularphase were significantly larger than those in the late luteal phase. Both insulin-like growth factor(IGF)-I and free fatty acid levels in the plasma were higher in the follicular phase than the luteal
phase. Estrogen treatment to ovariectomized goats increased the GH pulse amplitude, whereas
progesterone treatment decreased it. These observations suggest that the pulsatile pattern of GH
secretion in female goats varies with sex steroid levels and thereby affects IGF-I secretion andlipolysis during the estrous cycle. Although GH secretion was classically believed to be modulatedby the opposing actions of two hypothalamic peptides, GH-releasing hormone (GHRH) andsomatostatin (SRIF), recent studies suggest more complex mechanisms. When GH levels in the
peripheral circulation and GHRH, SRIF and neuropeptide Y (NPY) levels in the cerebrospinal fluid(CSF) in ovariectomized goats were simultaneously measured, there was a significant negative
cross-correlation and negative synchronicity between GH and NPY profiles, while there was nocorrelation between GH and GHRH or GH and SRIH profiles. In addition, intracerebroventricularinfusion of NPY suppressed GH secretagogue-induced GH release. We further observed that
estrogen treatment significantly decreased NPY levels in the CSF. From these observations, wecurrently hypothesize that the periodic decrease in NPY levels is involved in the generation of GH
pulses in goats, and that the modulation of GH pulsatility by sex steroids is also mediated at least
partially by NPY.

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Factors affecting successful implantation and maintenance ofpregnancy at the maternal-fetal interface in pigs
Heewon SEO, Yohan CHOI, Mingoo KIM and Hakhyun KA*
Department of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea*E-mail: hka@yonsei.ac.kr

Implantation process requires a well coordinated interaction between the maternal uterus andthe developing embryo in pigs. Embryo implantation in pigs occurs on around Day (D) 12 ofpregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes
various biological products such as estrogens, proteases, and interferons. Estrogens produced byconceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogenaction is explained by the endocrine and exocrine theory. The uterine endometrium becomes
receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells andincreasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone,progesterone, but the presence of conceptus in the uterus also induces changes of endometrial
functions, including most importantly maternal recognition of pregnancy. Many factors, such as
hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to
be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs.
However, understanding of the cellular and molecular events occurring in the endometrium is notcomplete. In a recent study that analyzed genes expressed in the endometrium of D12 of pregnancy
comparing with those of D12 the estrous cycle to identify the genes that were differentiallyexpressed and some subsequent studies, we made observations that some genes might be critical forthe establishment and maintenance of pregnancy. Firstly, we found that calcium regulatory
molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during
pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo
implantation and the maintenance of pregnancy. Secondly, we observed that salivary lipocalin(SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time ofembryo implantation, and its expression was regulated by estrogen. Thirdly, we found thatlysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of
implantation and LPA receptor 3 was uniquely expressed in the endometrium during earlypregnancy. Further analysis of those molecules will provide insights into the cellular and molecularbasis of maternal-and fetal interaction during pregnancy in the pig. More details will be discussed in
the presentation. [This work was supported by the BioGreen 21 Program (#20070301034040 and#20080401034003), Rural Development Administration, Republic of Korea]

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Neuroendocrine control of the pulsatile GnRH/LH secretion inruminants
Satoshi OHKURA1*, Yoshihiro WAKABAYASHI2 and Hiroaki OKAMURA2
1

Laboratory of Animal Production Science, Graduate School of Bioagricultural Sciences, Nagoya University,

Nagoya, Japan.
2

Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Japan.

*E-mail: saohkura@agr.nagoya-u.ac.jp

Pulsatile release of gonadotropin-releasing hormone (GnRH) is indispensable to maintainnormal gonadotropin secretion, which in turn regulates the gonadal activity in mammals. The
pulsatile secretion of GnRH is associated with synchronized electrical activity in the medial basal
hypothalamus— multiple unit activity (MUA)— which is thought to reflect the rhythmic
oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion, the GnRH
pulse generator. However, the cellular source of this ultradian rhythm in GnRH-releasing activity is
totally unknown. The aim of the present paper is to provide an overview of our current
understanding of the GnRH pulse generator, the longstanding "black box" mechanism, in goats. Our
main focus is to identify where is the location, and what are the components of this hypothalamicmechanism that governs pulsatile release of GnRH.
Kisspeptin (also known as metastin) is the endogenous ligand for the G protein-coupled
receptor, Kiss1r (now the term Kiss1r was assigned for the non-human kisspeptin receptor), andthere is growing evidence that kisspeptin/Kiss1r system in the brain is involved in the control ofreproductive functions. In the hypothalamus of male goats, kisspeptin -immunoreactive cell bodies
were exclusively found in the arcuate nucleus (ARC), the neural substrate implicated in theneuroendocrine control of GnRH pulses, suggesting that hypothalamic kisspeptin plays animportant role in the regulation of GnRH release in this species. Thus, direct input from the ARC
kisspeptin neurons to GnRH cell bodies in the medial preoptic area (MPOA) or their terminals in
the median eminence could be the intrinsic source for driving the GnRH pulse generator. To
determine whether the kisspeptin signalling could be responsible for producing pulsatile GnRH
secretion, we studied male goats, measured plasma levels of luteinizing hormone (LH) and recorded
MUA aimed at the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located.
Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in theposterior ARC, where kisspeptin neuronal cell bodies were found. Exogenous intravenous
administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing
MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of
the network of GnRH neurons— and could plausibly reflect the pacemaker activity of kisspeptin
neurons, whose projections reach the median eminence where GnRH fibers project. Theseobservations suggest that the kisspeptin neurons in the ARC may be the intrinsic source of the
GnRH pulse generator.
8

This work was supported in part by the Program for Promotion of Basic Research Activities forInnovative Bioscience (PROBRAIN) of Japan.

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Developmental potential and reprograming efficiency of porcine
embryos cloned with mesenchymal stem cells
Gyu-Jin RHO*
College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea*E-mail: jinrho@gnu.ac.kr

Errors leading to inappropriate expression could arise at any of the levels at which regulation ofgene expression occurs, including the organization of the nucleus, chromatin structure and theavailability of regulatory molecules. Studies on cloned embryos during preimplantation
development have revealed striking defects to faithfully recapitulate many of the essential early
events including transcription/translation, epigenetic modifications such as DNA methylation,
histone acetylation and chromatin configuration as well as genetic imprinting. In support of theseobservations, embryos produced by nuclear transfer (NT) consistently displayed a myriad ofdevelopmental abnormalities caused by aberrant expression pattern of genes involved in early
development, maintenance of pluripotency, epigenetic reprogramming and apoptosis regulation.
Altered levels of expression have also been detected in NT embryos when compared to their in vitro
and in vivo counterparts. However, the amount of information gathered on the profile of gene
expression in porcine NT preimplantation embryos is still scarce and limited to a handful of genes.
One of the factors in determining the efficacy of NT might be the type of donor cells. The oocytes
reconstructed from embryonic stem (ES) cells gave rise to an increase in the number of viable
offspring compared with somatic cells in mice, and a less differentiated cell type may support
greater development of NT embryos compared with terminally differentiated cell types. The nucleusof an undifferentiated cell may require less reprogramming for animal cloning than the genetic
material of a differentiated somatic cell. Hence, a comparison of efficacy among donor cells
between undifferentiated and differentiated mesenchymal stem cells (MSCs) would be interesting to
improve the efficiency.
We found that MSCs as donors in NT offer a greater potential to enhance blastocyst formation,higher total cell number and low incidence of apoptosis compared to somatic cells. Alterations in
the expression pattern of genes implicated in transcription and pluripotency (Oct4, Stat3, Nanog),
DNA methylation (Dnmt1, Dnmt3a), histone deacetylation (Hdac2), growth factor signaling andimprinting (Igf2, Igf2r) and apoptosis (Bax, Bcl2) regulation were observed in NT embryos. The
expression of transcripts in MSC-NT embryos more closely followed that of the in vivo derived
embryos compared with FF-NT embryos. The in vitro developmental ability of NT embryos derived
from undifferentiated MSCs was higher than those from differentiated MSCs or somatic cells.
In conclusion, MSCs with a relatively undifferentiated genome might serve as suitable donors
that could be more efficiently reprogrammed to re-activate expression of early embryonic genes in
porcine NT.

10Renewal of techniques related to embryo transfer in cattle – a novel
superovulation method using aluminum hydroxide gel
Koji KIMURA1*, Shuichi MATSUYAMA1, Hisataka IWATA2, Makoto SEKI3
and Makoto HIRAKO1
1

National Institute of Livestock and Grassland Science, Nasushiobara, Japan.

2

Tokyo University of Agriculture, 1737 Funako, Atsugi, Japan.

3

Kawasakiseiyaku K.K. 3-19-11, Nakaze, Kawasaki-ku, Kawasaki, Japan.

*E-mail: kimurak@affrc.go.jp

In the past two decades, embryo transfer (ET) of cattle has become a commonly used
reproductive technique in Japan. However, the pregnancy rate of ET has never been improved, as it
has constantly approximated 50%. On the other hand, many new findings and innovations were
established during this period not only in reproductive biology but also in machine engineering andmaterials science. Over the next decade, ET and its related techniques should be reconsidered andimproved using these new findings with the aim of increasing the pregnancy rate of ET.
According to the latest investigation, approximately 20,000 calves are produced using ET every
year in Japan. Although it is possible to use IVF embryos for ET, more than 80% of ET calves areproduced using in vivo-derived embryos. In order to obtain high numbers of in vivo-derived
embryos, superovulation (SOV) is usually induced in which FSH is administered to donor cattle ina step-down regime for 3 to 4 days to induce multiple follicular growth and ovulation. This
conventional SOV method is both time and labor intensive, and moreover, it can cause great stressin the cattle.
Recently, various types of adsorbants for macromolecules such as proteins or complex
medicines have been developed, allowing the chronic release of these macromolecules into theblood stream. Aluminum hydroxide gel (Al-gel) can adsorb macromolecules such as proteins or
toxoids and release them gradually. Accordingly, Al-gel is extensively used as an adsorbent or
adjuvant for vaccines. Therefore we investigated whether Al-gel could adsorb and release FSH
effectively in vitro and in vivo, and whether a single administration of FSH in Al-gel could
successfully induce SOV in cattle.
In the first experiment, it was investigated whether aluminum hydroxide gel could adsorb FSH
and release it in the presence of BSA, which is a major protein of interstitial fluid. When the gel
was mixed with FSH and centrifuged, the supernatant was recovered. Then the precipitated gel was
re-suspended in PBS containing 1% BSA. The solution was centrifuged, and the supernatant wasrecovered again. The concentrations of FSH in both supernatants were measured by RIA. Almost allof the FSH mixed with the gel was absorbed (99.9%), and more than 70% of the absorbed FSH wasreleased in the presence of BSA, which is a major protein of bovine interstitial fluid.
The aim of the next experiment was to identify whether a single injection of pFSH mixed with
Al-gel could induce multiple follicles to grow, ovulate and ultimately produce viable embryos and11to compared this result with that of the conventional 4-day serial injection method. Japanese Blackcows were given single injections of 30 mg (AU) pFSH with 5 mL Al-gel (3mg Al/mL)
intramuscularly (n=10) or multiple injections of pFSH (control; n=13) at 12 hr intervals for 4 days
(6, 6, 4, 4, 3, 3, 2 and 2 mg in saline). Estrus was induced in all cows by using 750 μg ofcloprostenol, and the animals were inseminated. The resulting embryos were recovered andclassified according to IETS guidelines. At the same time, blood samples were collected from someof the treated cows at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84 and 96 h after the initiation oftreatment to measure the concentration of FSH in plasma. The mean numbers of CLs and collected
and transferable (grades 1-2) embryos were 12.3 ± 2.7, 10.0 ± 2.5 and 8.6 ± 2.3/cow with the Al-gel
single administration versus 11.7 ± 1.8, 9.3 ± 1.7 and 8.0 ± 1.8/cow with the standard multiple
administrations, respectively. No significant differences were found between the two methods forany of the measurements. In the case of the single administration of pFSH in Al-gel, pFSH was notdetected in the circulation during the first 2 h after administration. The subsequent concentration ofpFSH rapidly increased to a peak at 12 h post-administration and gradually decreased thereafter. On
the other hand, in the case of multiple administrations, pFSH was detected in the peripheral bloodstream immediately after the start of treatment, reached a peak at 6 h and then decreased.
Al-gel is widely used as an adjuvant for vaccinations. Therefore, it was anticipated that
repeated administrations of pFSH with the gel would induce immune responses against thishormone and that, consequently, the cattle would not respond to SOV treatment again. Cows were
subjected to five successive SOV treatments at 2- to 3-month intervals over a year. However, thesuccessive SOV treatments using Al-gel did not result in a significant decrease in the number ofCLs, recovered eggs or recovered transferrable embryos among the SOV periods.
Our results suggested that Al-gel could adsorb and release pFSH effectively in vitro and in vivo
and that a single administration of FSH in Al-gel could successfully induce SOV in cattle.

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The ovary, as the source of stem cell
Jeong Mook LIM*
Major in Biomodulation/WCU, Department of Agricultural Biotechnology, Seoul National University, Seoul,
Republic of Korea. Research Institute for Agriculture and Life Sciences, Seoul National University Hospital,
Seoul, Republic of Korea*E-mail: limjm@snu.ac.kr

It has generally been accepted that the utilization of the stem cells that have either multipotent
or pluripotent activity is the key factor for developing cutting-edge technologies for cell
replacement, organ regeneration, tissue engineering and rejuvenation therapy. Extensive andcontinuous efforts have been made to establish histocompatible stem cells to date. At the very earlystage, a technology to transfer a somatic cell into an oocyte being enucleated, named somatic cell
nuclear transfer (SCNT), was suggested for the derivation of patient-specific stem cells (1).
However, R & D activity on SCNT definitely accompanies with sacrificing of huge amount of
viable oocytes and embryos, which incurs great concern on abusing of human oocytes, as well as
the concern on reproductive cloning of the human (2,3). To overcome such limitation,parthenogenetic activation of human oocytes, interspecies nuclear transfer of human somatic cells
into animal oocytes and alternative technique of nuclear transfer for generating unimplantableembryos have been subsequently suggested (4-6).
We have additionally sought for other alternatives and our effort has focused to develop
clinically feasible technology with minimizing of ethical issue. We have selected the research using
ovarian tissue based on our previous experience of research. Considering on general procedure of
drug discovery and development, however, the boundary of our research has restricted within
animal model experiment at present. We have attempted to derive histocompatible ESC frominactivated germline cells. In mammalian ovaries, there exist numerous immature follicles called
preantral (primordial, primary and secondary) follicles in the ovaries. In one’s human life,
extremely small number of follicles, which is as less as 1% of total number, can develop into theGraafian follicles releasing developmentally competent, mature oocytes into the fertilization site (7).
Most immature follicles gradually disappeared after birth and except for mobilized follicles, the rest
remaining ‘developmentally dormant’ in the ovarian cortex or medullary tissues finally became
degenerated via apoptosis. There has been no attempt to establish histocompatible embryonic stem
cell (ESC) by culturing of preantral follicles, which avoids the use of developmentally competent
oocytes for stem cell establishment. We, therefore, have attempted to develop a technique for invitro-culture of preantral follicles. To retain autologous ESC, parthenogenetic activation of oocytes
matured in in vitro-cultured follicles was employed.
As results, we established histocompatible stem cells from the culture of preantral follicles atthe secondary stage. Also, we can derive histocompatible ESC and viable blastocysts from adultovarian tissue and the culture of primary follicles. Follicle bank system was established by13

comparison of different freezing protocols. A technique for preantral follicle culture can be
employed for reproductive medicine and cancer therapy and considerable number of preantral
follicles can be retrieved from the patients suffering from the infertility of uterine factor and fromthe cancer patients scheduled for chemotherapy. Accordingly, the preantral follicle culture
contributes to developing various therapeutic technologies.
References
1. French AJ, Adams CA, Anderson LS, Kitchen JR, Hughes MR, Wood SH. Development of
human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts. StemCells 2008;26:485-493.
2. Taupin P. Stem cells engineering for cell-based therapy. J Neural Eng 2007;4:59-63.
3. Mertes H, Pennings G. Oocyte donation for stem cell research. Hum Reprod 2007;22:629-634.
4. Kim K, Lerou P, Yabuuchi A et al. Histocompatible embryonic stem cells by parthenogenesis.
Science 2007;315:482-486.
5. Chang KH, Lim JM, Kang SK, Lee BC, Moon SY, Hwang WS. An optimized protocol of ahuman-to-cattle interspecies somatic cell nuclear transfer. Fertil Steril 2004;82:960-962.
6. Meissner A, Jaenisch R. Generation of nuclear transfer-derived pluripotent ES cells fromcloned Cdx2-deficient blastocysts. Nature 2006;439:212-215.
7. Moore KL, Persaud V. Before we are born; Essentials of embryology and birth defects, 6th ed.
Philadelphia: Saunders, 2003;10-26.

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A novel concept for the bovine luteolytic cascade with a focus on luteal
blood flow and vasoactive factors
Koumei SHIRASUNA1*, Motozumi MATSUI2, Takashi SHIMIZU1 and Akio
MIYAMOTO1
1

Department of Animal and Food Hygiene and 2Department of Clinical Veterinary Science, Obihiro

University of Agriculture and Veterinary Medicine, Obihiro, Japan*E-mail: shirasuna@obihiro.ac.jp

The corpus luteum (CL) undergoes drastic changes in its function and structure during the
estrous cycle. If pregnancy does not occur successfully, luteolysis is caused by prostaglandin F2α(PGF2α) released from the endometrium around days 17-19 of the estrous cycle in the cow. During
the past 30 years, it has been proposed that a rapid decrease in luteal blood flow is one of theessential impacts of PGF2α. As the CL matures, the steroidogenic cells establish contact with many
capillary vessels, so that the CL is composed of a large number of vascular endothelial cells that canaccount for up to 50% of all cells in the bovine CL. Also, luteal endothelial cells secrete several
vasoactive substances such as PGF2α, nitric oxide (NO), endothelin-1 (EDN1) and angiotensin II(Ang II). Therefore, blood vessels and endothelial cells within the CL should have an essential rolein luteal function in the cow, in which the vasoactive molecules above may regulate luteolysis
locally.
Luteal blood flow: Recently, we discovered that a PGF2α treatment of cow having the mid CL (Day
10 of the estrous cycle), but not the early CL (Day 4 of the estrous cycle), induced an acute increase(0.5-2 h) in luteal blood flow at the periphery of the CL. During spontaneous luteolysis in the cow,
an increase in blood flow in the periphery of the CL was associated to peak of plasma PGFM (aproduct of the metabolism of PGF2α), just prior to the decline in P4 secretion, strongly suggestingthat PGF2α from the uterus induces an acute blood flow increase in the CL.
NO and luteal blood flow: NO, a strong vasorelaxant, could be a good candidate in the blood flow
increase. Consistent with the luteolytic ability of PGF2α, PGF2α stimulated the expression ofendothelial NO synthase in the periphery of the mid CL after PGF2α administration but not in theearly CL. In the peripheral area, resistance vessels (>20 µm) having vasomotor function localized
mainly in the mid CL, but they were not observed in the early CL. A direct injection of the NO
donor (SNAP) into the mid CL clearly induced an increase in blood flow, and the injection of the
NOS inhibitor (L-NAME) completely suppressed the increase in luteal blood flow induced by
PGF2α.
Vasoactive factors: Luteal EDN1, Ang II and PGF2α are involved in the process of luteal regression.
Using in vivo microdialysis system for the bovine CL, intraluteal EDN1, Ang II and PGF2α
secretion increased after onset of spontaneous and PGF2α-induced luteolysis. For the luteal function,
EDN1 and Ang II inhibited P4 secretion in luteal cells. Moreover, the intraluteal EDN1 or Ang II
injection after 1/4 dose of PGF2α administration (sub-luteolytic dose) resulted in a reduction of15

plasma P4.
On the basis of the results of the series of experiments, the following scenario can be drawn forthe luteolytic cascade in the cow (Fig. 1). Endogenous (from the uterus) or exogenous PGF2α
directly activates eNOS/NO in the peripheral area of the CL, so that strong vasorelaxant NO isdrastically produced in periphery of the CL especially large luteal vascular vessels. Therefore, luteal
blood flow in the peripheral area are increased acutely by the vasodilative action of NO, suggestingthat PGF2α-induced increase in luteal blood flow is one of the earliest physiological events in theluteolytic cascade in the cow. Following an increase in luteal blood flow, CL-derived PGF2α now
stimulates production of ET-1, Ang II and luteal PGF2α. These vasoactive substances decrease theP4 secretion. After this stage, these vasoactive molecules are kept at high levels in the CL andinduce vasoconstriction to shut-off the blood supply to the CL.
In conclusion, the bovine
CL is a large and heterogeneous
endocrine organ, and PGF2α hasasite-restrictedactiondepending not only on the luteal
phase (i.e., early vs. mid) but
also on the region of the CL. A
three-dimensional
vascularstructure in the CL andinteractions among several
vasoactive factors are required
for maximal responsiveness to
PGF2α to achieve a rapid
regression of the CL in the cow.

16

Contact persons:Kazuhiro KIKUCHI
National Institute of Agrobiological Sciences, Japan
E-mail: kiku@affrc.go.jp
Yong-Mahn HAN
Korea Advanced Institute of Science and Technology, Republic of Korea
E-mail: ymhan@kaist.ac.kr

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