TPC NEWS Vol. 5, No.2,
Fall, 1986 (Whole Number 10)
(
English Summary )
Page
-3 Essay : My impression of three primate research institutes in Japan
In
Japan, there are several research institutes for studying nonhuman primates.
But they are not generally known to the public, because of their
specialty in studying. Among those
institutes, I think, however, Japan Monkey Center (JMC) in Inuyama, the Primate
Research Institute of Kyoto University (PRI) in Inuyama and our center (TPC) in
Tsukuba are relatively well known. These three institute have some
characteristics in common, but also have different characteristics derived from
their own history and purposes.
By the way, I make a round trip between Inuyama and Tsukuba at least
several times a year. At this moment, I am writing this manuscript in a Shinkansen
(bullet) train on my way to Inuyama. For
a long time I have attended at study meetings held under the auspices of JMC.
Until several years ago, I had been a member of the Editorial Board of
Primates which is a scientific journal published by JMC.
It has been a great pleasure for me to attend these meetings and to know
fruits of studies in various fields of the primatology.
And now, I often have opportunities to visit the PRI to attend the
meetings of the governing board of the Japan Primate Society which was
established last year. These
experiences have given me vivid impressions concerning the properties of these
institute.
JMC has the longest history among them.
It has performed a great number of scientific works widely ranging over
various research realms. Many staff
members who had been raised in this center have joined and contributed greatly
to the establishment of the PRI. Today,
JMC is specifically making international contribution to the development of
primatology by publishing the journal, "Primates", as well as
by keeping a zoo and a museum.
As for the PRI, I feel it is a place where various recondite studies are
conducted in the atmosphere of traditional academism and in the reformative
spirit of young researchers. The
most essential conditions for researches, such as freedom, independence and
criticism are almost perfect there. PRI
is just a typical "sanctuary" for the primatological studies in Japan,
I think.
Well, what impression do they have on our center, TPC?
TPC's history is only eight years old or so.
Its bases is not fully established yet.
But different from the other two institutes, its purpose is clearly
defined. It can be said that TPC is
an institute at which mission-oriented researches are conducted. Practical studies to solve various problems occurring in
daily breeding and rearing works, basic researches to elucidate biological
characteristics of nonhuman primates and investigations to find out the
usefulness of nonhuman primates as laboratory animals are actively being done
there.
I believe, in any case, if these three primate research institutes grow
while promoting their own characteristics and make active interchange of
researchers, it would be very useful for the progress of primatology in Japan.
Page-4
Breeding Topics: Two twin cases - Rearing report II
I reported about two twin-birth cases of cynomolgus monkeys in the former
issue, Vol. 2, No. 3, 1983. The
cases are now four and three years old, respectively.
This time. I'd like to
present you their subsequent record of rearing.
The first twins (both females) were weaned from their own and foster
mothers at about 6 months of age. After
they lived together in the same cage, they were grouped with other three females
and a male of about the same age. Their
weight growth was normal as shown in the figure.
Shortly after they became two years old, one of them fractured her tail
at the point of 15 cm from the end. The
tail was cut surgically. Then the
monkey broke her second finger of the right hand one year after the tail
fracture. Moreover she got hurt badly on the second finger of her left
hand after three weeks. Generally,
injuries and fractures like these are sometimes seen in colony-bred monkeys,
since infant monkeys enjoy fingering narrow spaces and holes.
So, we have to reform inner surface of the cage for infant monkeys.
Now, the twins have sexually matured.
We observed the first menstrual bleeding in one of them in March this
year. She was three years and ten
months old. The average age of the
occurrence of the first menstruation in the cynomolgus monkey of this center is
two years and 6-10 months. We hope that both of them will be pregnant simultaneously.
The second twin babies (both females) were born by Caesarean operation.
One of the pair had the lowest birth weight(138g) so far.
She was nursed by a foster mother for about eight months.
During this period, she broken her tibia of the right leg.
The fracture was not so serious that she recovered in a month without any
operation. At the weaning time, her
weight was 692g, while the other weighed 938g.
Then the twins were made to live together in a cage, although there was a
much difference in weight (Usually, the weight difference between peer mates
kept in a cage at TPC is within 50g.
Four months later they were put into a group feeding cage together with
another male and female. Their
weight were 1230g and 1030g at that time, respectively.
One month later, the group was broke up, since bullying happened. Another new group was made, but it resulted badly, too.
The third group consisting of four monkeys was made.
Now it is going well. We
hope these twins will serve for the breeding and experiments in which their
biological characteristics are efficiently utilized.
Page-5
A Survey of Campylobacter Infection in the Cynomolgus Monkeys Recently Imported
The data we obtained with 385 cynomolgus monkeys imported from the
Philippines between September in 1983 and October in 1985 are shown in the
tables.
Page-5
A Lecture by Dr. T. C. Jones (New England Regional Primate Research Center)
A lecture was presented by Dr. T.C. Jones, Ex-director of New England
Regional Primate Research Center (NERPRC) and professor emeritus of Harvard
University, at TPC on March 24, under the auspices of the Japanese Working Group
for U.S.-Japan Science Cooperative Program-Laboratory Animal.
It was also supported by the Corporation for Production and Research of
Laboratory Primates and TPC's Researchers Association.
The title was The New Regional Primate Research Center.
Dr. Jones presented his lecture for about one and half hours, explaining
clearly and comprehensively the history and current situations of the NERPRC.
Especially, it was our surprise to hear that the NERPRC has several times
as many scientists and technical personnels as TPC has, although the number of
monkeys kept at NERPRC is less than that at TPC.
After the lecture, the audience asked him some concrete and live
questions and he kindly answered them. We
all attending the lecture had the impression that the exchange between Japan and
America in this field had taken a step forward.
Page-6
Introduction of Facilities: A radio isotope (RI) laboratory
Use of radio isotope (RI) is now indispensable in the fields of medical
science and biology. In this
center, an unsealed RI is used to elucidate various physiological
characteristics of nonhuman primates. The
floor area of our RI facilities is 76 m2, consisting of two
laboratories, a room for measuring apparatuses, a test room of radioactive
contamination, and RI storage. A
treatment room of RI drainage (16 m2) is also provided as an annex.
Main works carried out here are measurements of various hormone levels in
the blood of nonhuman primates and the development of new determination methods.
Usually, a radio immuno assay (RIA) system using 125I and 3H
is performed to determine follicle stimulating hormone (FSH) levels.
A new radio- receptor assay (RRA) system to determine serum luteinizing
hormone (LH) level was established in this facility.
Physiological characteristics of ovarian LH-receptors and immunological
characteristics of nonhuman primates have been analyzed by using 3H
or 51Cr.
This facility was designed to treat a lot of samples simultaneously
without requiring many hands. Samples
and reagents are automatically put into small test tubes and centrifuged.
In the RRA system, plastic centrifugation tubes (1.5 m1) are used.
After the end of incubation the tubes are immediately centrifuged at
6,000 rpm for 4 min by using a rotor which can treat 64 tubes (1.5 m1) or 100
tubes ( 0.2 m1 and 0.4 m1) at a time. Using
a liquid scintillation counter results in a large quantity of RI drainage.
So we use mini-vials (2 m1) not to make much RI drainage.
But a considerable amount of drainage has been accumulated for the past 7
years. The opening of the treatment
system of RI drainage is expected in this year.
This RI facility may be the smallest one situated in Tsukuba Science
City, but it is contributing greatly to the study of physiological
characteristics in nonhuman primates and is used very efficiently by promising
young researchers.
Page-7
On the Techniques of Care and Management of Cynomolgus Monkeys:
An attempt to decrease the incidence of injured and diarrheal cases in
weanling cynomolgus monkeys----- Nursing-mother system----
TPC got 1749 normally born cynomolgus monkeys from the beginning of 1979
to the end of 1 985. Their weaning
rate was 96%. Weaning is performed
more than 90 days after birth, when infant monkeys grow to be over 700 g in
weight, develop the first deciduous molar and can get diets by themselves. Then they are tattooed identification numbers.
In our previous system, weaned infants were reared as follows:
Every two infants were
kept together in a cage for more than three months.
Then, four to eight peer mates were reared together as a group until they
sexually mature. During this rearing period, however, many cases of diarrhea
and injury were found. Much time
was wasted to cure those cases.
Thus, we have tried to adopt an improved rearing system in which nursing
mother monkeys are used. An adult
female monkey (nursing mother) is made to live with four infants in a cage for
the first one month after weaning. Nursing
mothers employed do not have any kinship with the infants but experience of
maternity.
As the result, the incidence of diarrheal cases occurring just after
weaning decreased to one fourth of that in the previous rearing system.
Injury cases were hardly seen either even after nursing mothers were
separated. But slight wounds given
by a nursing mother were noted in some infants.
To prevent these wounds, we have to make further studies on selecting
good nursing mothers.
Page-8
Case Report.: Salmonellosis in a cynomolgus monkey
Salmonellosis is one of the important infectious diseases which mainly
affect gastro-intestinal tracts in both man and animals.
Generally, the pathogenicity of Salmonella organisms seems to be low in
animals, especially in infants. Our
bacteriological survey performed recently with 130 cynomolgus monkeys imported
from the Philippines in 1 985 indicated that 7 animals were positive for Salmonella (s. typhimurium) at the time of arrival.
Four out of the seven animals died 5,15,17 and 22 days after arrival,
respectively. Every death-case had
suffered from serious malnutrition.
History
of the case: Five or more years old (estimated age) female cynomolgus monkey
of Philippine origin. Three days
after arrival, she excreted watery stool. The
watery stool changed to bloody stool two days later.
Twenty five days after arrival, she fell into a moribund state and was
sacrificed. She had showed diarrhea
all through this period but no activity and appetite.
Chloramphenicol, cefazolin, refampicin and glucose solution were
administered.
Autopsy
findings: Serious loss in weight. Mucous membranes of the caecum and colon were distended, and
a light edematous swelling was noted in them.
The size of lymphnode was normal. Contents
of the large intestine were pale yellowish paste.
The stomach and small intestine showed almost no gross abnormality.
Histopathological
findings: In the small intestine, the adhesion of villi, epithelial
degeneration and separation and slight hyperaemia of lamina propria were
recognized. In the large intestine,
the epithelial tissue was degenerated and separated, The lamina propria was
infiltrated with many small round or hitocyte-like cells.
Hyperaemia and edematous swelling were observed in the mucous membrane
and the submucous layer. Leucocytes
were found in a part of glandular cavity. This
case had not any abnormal findings which seemed to be caused by Salmonella
infection in other internal organs.
Page-9
A Comment on My Research - Physiological characteristics of ovarian LH-receptors
in cynomolgus monkeys
Female cynomolgus monkeys reach puberty at the age of about two to three
years. Their menstrual bleeding is
observed for 1 -5 days, and its cycle is 29. days on the average.
Their menstrual mechanism is very similar to that in humans.
Therefore, studying ovarian functions in cynomolgus monkeys is necessary
and useful for the better understanding of human female reproduction.
Usually, ovarian functions are analyzed in relation to serum gonadotropin
concentrations during menstrual cycle. Typical
pattern of changes in serum gonadotropin level measured by RIA assay method is
shown by Figure 1. In this scheme,
no marked increase of serum LH is observed at the stage of active luteal
function. However, when we use the
radioreceptor assay (RRA) method established in our laboratory, the increase of
serum LH was observed also at this period (Fig. 2) , though the number of the
tested cases was not so many. This
fact suggests that the following possibility: the Immunoreactive LH measured by
the RIA might be not the same substance as the LH(receptor binding) measured by
the RRA method.
Protein hormones such as LH and FSH are carried with blood stream to
their target organs then they are bound to hormone receptors on the cell surface
of the organs. Hormones can not act
without binding to hormone receptors. In
other words when LH affects the ovary, the presence of ovarian LH receptor is
essential. So, the specific binding of LH should be scrutinized.
An experiment to detect the specific binding of ovarian LH receptor was
carried out as follows: Six pooled ovaries (-80 C) from three cynomolgus monkeys
of wild origin were used as experimental materials.
A total amount of LH bound to ovarian tissue was determined following the
reaction of 125I-LH to ovarian samples.
Next, the mixture of 125I-LH
and a large quantity of non-labelled hormone was reacted to ovarian samples to
measure an amount of nonspecific binding.
Specific binding of 125I-LH was calculated as follows: (Total
binding)-(Nonspecific binding) = (Specific binding)
From this experiment, 7 mg ovarian tissue per tube were judged to be
sufficient. Then we examined the
presence of LH receptor in the ovary and the characterization of physiologic
properties of LH receptor during menstrual cycle.
We planned this experiment, supposing that the characteristics of LH-
receptor would markedly change at the time of ovulation after which ovarian
function changes into luteal phase. But
this hypothesis was quite incorrect. Specific
binding of LH was hardly observed in the ovaries without maturing follicles
(Fig. 5) as well as in the ovaries with matured follicles (Fig. 6). And a marked increase of the specific binding was observed
only in the ovary with a large corpus luteum (Fig. 7). Then we confirmed the reason for this marked increase of
specific binding using two ovaries of the cynomolgus monkeys whose menstrual
cycle was on day 26. This
examination revealed that the 125I- LH specific binding was on
account of the large corpus luteum.
The number and affinity constant of ovarian LH-receptor was also able to
be determined from the same data (Scatchard plot method).
The result obtained from 17 female monkeys with different phase of
menstrual cycle are shown in the bottoms of' Fig.
5,6 and 7.
The hormone receptor at the follicular phase and ovulatory period showed
low affinity, while LH-receptor at the luteal phase had high-affinity.
Thus, it was clearly demonstrated that the ovarian LH-receptor in
cynomolgus monkeys changed dynamically in its characteristics.
The susceptibility of ovaries to LH in both ovulatory and preovulatory
phase is very small, while the ovary in luteal phase is very susceptible to LH.
Therefore, serum LH level
in ovulatory and preovulatory phases is much higher than that in luteal phase.
Further detailed studies on the serum LH concentration during the luteal
phase are needed.
By the way, the studies on testicular LH-receptor in rhesus and green
monkeys have been reported from other laboratories.
In the testes of these primate species, only LH-receptors similar to
those of low affinity in the ovary seems to be existing.
Furthermore, it is said that human ovarian LH- receptor was found only in
the luteal phase. This fact might be corresponding to the presence of the high
affinity in the cynomolgus monkeys. In
this regard, it can be stated that the cynomolgus monkey is an excellent animal
model.
Page-12
A Special Article: I expect much from TPC
In March, I visited the Southwest Foundation for Research and Education
in the U.S. to attend a symposium on primate genetics.
Then in July, I attended an international symposium of veterinary
immunology held in Guelph University, Canada.
In these trips I had a chance to visit some primate research centers and
The Jackson Laboratory in the U.S., These opportunities made me think over the
perspective of medical and biomedical researches using nonhuman primates.
It was 1974 when I went to the U.S. and Europe to observe primate
research centers prior to the start of the plan of establishing TPC.
In those days, it was urgently needed in the U.S. to establish the
domestic breeding system of nonhuman primates, owing to the total ban of export
of rhesus monkeys by the government of India.
Originally, primate research centers in the U.S. were established, having
close relation with the departments of medicine and veterinary medicine of
universities. So the research centers have been the places where many types
of studies are conducted by the researchers of the centers own and by those from
universities. The main activities
of those research centers were to do research itself on medical and biomedical
science, and breeding activities were limited except unavoidable cases such as
studies using newborns.
Almost all domestic
breeding programs schemed in the U.S. during the past decade on account of the
shortage of laboratory primates adopted the system of large- scale group
breeding mainly performed in out-door colonies.
Today those breeding programs are going well.
But there is still a tendency that researchers prefer using the monkeys
of wild origin which may be got cheaper.
On the contrary, TPC, was established to breed laboratory primates of
good quality under the strictly artificial conditions.
I think, there is not yet any other large-scale breeding facility like
this in the world. In the field of breeding, TPC may be ahead of most American
primate research centers. But oh
research aspect, it is far from the primate research centers in America because
of the organizational limitation.
The importance of the studies using nonhuman primates is more and more
increasing in every field of medical science. For example, the analysis on
molecular basis of major histocompatibility antigen system has become possible
also in nonhuman primates. AIDS
problem has brought about a rapid advancement in the immunological analysis of
nonhuman primates as a model of AIDS patients, At all events, the future of
laboratory primates as an animal model is much expected, especially, regarding
serious human diseases such as Parkinson's disease as well as human reproductive
problems like in vitro fertilization and so on.
Now, TPC has become a very competent breeding center that can supply good
laboratory primates being suitable to those researches.
I think, its further successful development is depending on how TPC
establishes its research system.
The Jackson Laboratory I visited this time, is known as an institute
which has been making a contribution to the medical and biomedical sciences
through developing and supplying mice of good quality. It also opens the doors
for the researchers all over the country.
Being the first example in the world, TPC has established an excellent
breeding system for laboratory primates. I
am expecting that TPC will play also the role of the primate research
center like The Jackson Laboratory.
Page-13
Overseas Topics Animal Testing: Why the chemical industry says it is
necessary
Japanese translation of the leaflet "Animal Testing: Why the
Chemical Industry Says it is Necessary" sent from Mrs. M.T.
MacMillan (Head of International Trade, Chemical Industries Association
Limited, London U.K.) is presented here as an material to think about problems
of animal testing and animal protection.
Page-14
Japan-China Joint: Workshop on gLaboratory Animalsh
Japan-China joint workshop on "Laboratory Animals"
was held at the Science and Technology Agency, Tokyo on March 19 and 20.
Four representatives from China, a secretary of science of the Chinese
Embassy and a student staying in Japan, and about 30 researchers from Japanese
side including the Chief of Life Science Section of the Science and Technology
Agency, attended.
This was the first meeting to be held for the researchers themselves
although the exchange through visiting each country was made last year.
The sound development of the exchange between Japan and China is very
expected. Next joint workshop will
be held in China.
Page-15
A Clinical Commentary by a Veterinarian: TPC Dental Clinic
One day a female cynomolgus monkey was bitten on her face by a male
breeder. The wound was pretty
serious. I shaved off her hair of
the face and back, and found several other wounds on the shoulder and arms.
I treated them and sutured the facial wound of 10 cm in length.
Well, the male who injured the female was the owner of very big canine
teeth. Those canine teeth must be
cut or pulled out. The method of canine extraction formerly performed at TPC was
primitive. All instrument needed
for the method was only cutting pliers. But this way sometimes caused bacterial
infection in dental pulp cavities.
I asked the doctor of Kanagawa Dental College to teach me the method of
canine teeth extraction which is carried out in human dental clinic.
First of all, I had to get a complete set of tools and materials required
for the dental treatment. At last,
the preparation for opening TPC Dental Clinic was ready.
Orthodox cutting and treating procedure takes real dental doctors about
30-40 minutes for four canine teeth, but it does me about one hour.
Two years has passed since I began canine teeth cutting by this method.
There has been no trouble in dental pulps so far.
But there is a long procession in front of TPC Dental Clinic because I, a
cub-dentist, need much time per a patient. .
Page-16
A Memory of a Trainee: My experience at TPC for a year
I clearly remember something experienced when I first entered an animal
room of TPC. It was about one and
half years ago. I had to change my
clothes thoroughly into working dress (uniform coveralls) and had to put on a
head cover, a mask and rubber boots. This
heavy outfit made me be aware of the danger or biohazardousness of using
wild-originated monkeys that are close relatives of man and may have many .kinds
of infectious agent transmissible to man. Since
that time, whenever I changed my clothes to enter animal rooms I am reminded of
this matter.
Many staff members were working with monkeys in well devised facilities
and by a perfect system of animal care and management.
I observed mutual behaviors between mother and infant cynomolgus monkeys
mainly in the room for rearing young cynomolgus monkeys.
At times I was threatened by mother monkeys, and twice was extraordinary
frightened at pretty strong earthquakes that occurred during my observation
time. But, I was very comforted at
the sight of innocent monkeys and used to have pleasant time with animal
technicians.
To observe behaviors required much concentration and attentiveness, even
a slight difference in daily routine seemed to influence infant monkeys.
Owing to kind consideration and warm cooperation of the staff members, I
could continue my behavioral study and completed my graduation thesis.
According to my observation, the process of separation between mother and
infant cynomolgus monkeys was as follows: Infants firstly separated from their
mothers, and came back to the mothers again.
The mothers accepted the infants within a certain period.
But after the period they rejected the infants coming near to them.
The separation of mother and infant monkeys was established through
repeating this pattern of mutual actions. In
addition, the separation was completed more definitely and rapidly :W; male
infants than in of females.
During my training period, what I deeply felt was that the importance of
the role of animal technicians who greatly supported researchers.
I would like to express my appreciation to Dr. Honjo and other staff
members, and of course, animal technicians who kindly guided and helped me.
Page-17
Sketches from Animal Rooms
Meeting
Again:
An animal-technician who had temporarily worked for another laboratory
for two years came back to TPC. He
met again the monkeys once he earnestly took care of.
But the monkeys did not remember him and made a big noise to see him.
He also was surprised to see not a few monkeys were overweight, though he
himself had gained weight, too. They
both had put on weight because of the lack of exercise.
He will be able to lose his weight by himself because he is free.
But what should the monkeys being kept in cages do?
I
am a freshman at TPC:
A fresh and young animal-technician is describing his impression on his
daily works, making a fresh determination to become a good animal-technician.