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In vitro fertilization analysis of squirrel monkey oocytes produced by various follicular induction regimens and the incidence of triploidy.

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American Journal of Primatology W.37-48 (1993)
In Vitro Fertilization Analysis of Squirrel Monkey Oocytes
Produced by Various Follicular Induction Regimens and
the Incidence of Triploidy
DONNA L. PIERCE', M. P. JOHNSON', J. B. KANEENEZ,AND W. R. DUKELOW'*2
%ndocrim Research Center, Michigan State University, East Lansing, Michigan; 2Center
for Population Medicine, College of Veterinury Medicine, Michigan State University, East
Lansing, Michigan
An in vitro fertilization system utilizing squirrel monkeys was used to
compare follicle-stimulating hormone, clomiphene citrate and prostaglandin E, as follicular induction regimens, analyze culture medium characteristics, and examine the physiological phenomenon of polyspermy. Induction of follicular growth was poor with clomiphene citrate when
compared to the control group and increased the incidence of atretic follicles a t all levels tested. When prostaglandin E, was administered, larger
numbers of mature oocytes were recovered at laparoscopy. There was no
difference in fertilization rate between the treatment and control groups.
Homologous serum was an adequate protein source in TC-199 fertilization medium for squirrel monkey oocytes. Although the rate of triploidy
was increased with in vitro fertilization, there was no relationship between sperm concentration and the incidence of polyspermy. These findings demonstrate that the squirrel monkey is a valuable primate system
for studies of in vitro fertilization and preimplantation development.
0 1993 Wiley-Liss, Inc.
Key words: in vitro fertilization, ovulation,squirrel monkeys
INTRODUCTION
Induction of ovulation in some species of old world monkeys was initially
reported by injecting extracts of monkey pituitaries and human menopausal gonadotropin (HMG) [van Wagenen & Simpson, 1957; Simpson & van Wagenen,
19621. The first experiments in new world primates were described by Bennett
[1967], who used injections of pregnant mare's serum gonadotropin (PMSG) and
human chorionic gonadotropin (HCG) to induce follicular growth and ovulation.
However, repeated administration of PMSG results in a refractoriness due to
PMSG antigenicity IOzasa & Gould, 19871.
Some years ago studies were undertaken to induce single or double ovulations
in the squirrel monkey [Dukelow, 19701. Initially monkeys were pretreated with 5
Received for publication September 6, 1991; revision accepted September 10, 1992.
Address reprint requests to W.R. Dukelow, Endocrine Research Center, Michigan State University, East
Lansing, MI 48824.
0 1993 Wiley-Liss, Inc.
38 I Pierce et al.
mg of progesterone for 5 days to mimic the luteal phase of the cycle. The animals
were then given various treatments of follicle stimulating hormone, HMG, and
PMSG. The optimal regimen consisted of four days of 1mg of FSH followed by a
single injection of 250-500 IU of HCG on the last day. Oocytes produced from
follicular induction regimens are capable of being fertilized both in vivo [Jarosz et
al., 19771 and in vitro [Kuehl & Dukelow, 1975b, 1979; Chan et al., 19821. It was
later found that the pretreatment was unnecessary.
Induction of follicular growth in the squirrel monkey has been achieved using
human urinary follicle-stimulating hormone (hFSH) [Yano & Gould, 19851. The
authors reported hFSH to be as effective as HMG for inducing follicle development
and superior to both HMG and porcine follicle stimulating hormone (pFSH) at
lower dose rates.
The involvement of prostaglandins either directly or indirectly in mammalian
ovulation has been shown in the rhesus monkey [Batta & Brackett, 1974; Wallach
et al., 1975; Batta et al., 19781. A consistent method for producing superovulation
in the rhesus monkey using a combination of gonadotropins and prostaglandin E l
was reported by Batta et al. [19781. Controlled ovulation has been achieved in the
marmoset (Cullithrixjacchus) with human chorionic gonadotropin and prostaglandins [Hodges et al., 19871, and oocytes have been fertilized in vitro and transferred
to produce a living young in this species [Lopata et al., 19881.
The area of in vitro fertilization and embryo transfer (IVF-ET) in nonhuman
primates has been reviewed [Dukelow, 1983a,b,c; Wolf et al., 19901. In vitro fertilization in the squirrel monkey was first announced in 1972 [Cline et al.; Johnson
et a1.1 with the first full publication from the former workers appearing in 1973
[Gould et al.]. Later studies achieved a 40% fertilization rate [Kuehl and Dukelow,
1975bl. Chan et al. [19821 showed that if ova collected 15-16 hours after HCG
injection were allowed to incubate an additional 21 hours before sperm introduction, levels of fertilization were increased. In addition, supplementation with 1
micromole of dibutyrl CAMPto the culture medium increased the in vitro fertilization rate from 60% to 90%.
METHODS
Animals
Adult squirrel monkeys (Suirniri sciureus) of Bolivian and Guyanan origin
(South American Primates, Miami, Florida) were housed on a 12:12 hour light:
dark cycle at a temperature of 21 3°C. During the summer months (June to
October), the animals were maintained in large colony cages outdoors [Jarosz &
Dukelow, 19761.
*
Follicular Induction Regimens
The females received one of the following follicular induction regimens: a) FSH
plus HCG (control), b) clomiphene citrate plus HCG, c) FSH, prostaglandin El plus
HCG, and d) prostaglandin El without HCG. The following protocol was used in
the control group: Female squirrel monkeys received four daily i.m. injections of 1
mg follicle stimulating hormone (FSH-P", Burns-Biotec Laboratories, Inc.,
Omaha, NE) and a single 250 IU, i.m. injection of human chorionic gonadotropin
(HCG) (A.P.L.@Ayerst Laboratories, Inc., New York, NY) on the fourth day
[Dukelow, 1970, 19791. Five daily FSH injections, rather than four, were given
during the anovulatory months (July through September), followed by HCG
[Kuehl & Dukelow, 1975al.
Clomiphene citrate was mixed with Gatorade" (Stokely-Van Camp, Inc.,
Follicular Induction / 39
Chicago, IL) and given orally a t doses of either 2 mg, 4 mg, or 6 mg per animal for
four days followed by an i.m. injection of HCG on the last day.
Prostaglandin El (2 mg, Sigma Chemical Co., St. Louis, MO)was tested by
administration of FSH for four days with an injection of prostaglandin Elon the
afternoon of the fourth day given either with or without an additional injection of
250 IU of HCG.
Laparoscopic Recovery and Culture of Oocytes
Follicular oocytes were obtained by laparoscopic techniques described previously [Dukelow, 19781 approximately 16 hours after HCG.
The ovarian follicles were counted according to size (large 3 mm; medium 3
mm to 1mm; small <1 mm) and aspirated using a 25 gauge 5/8 inch needle into
0.05 ml of TC-199 culture medium (with 25 mhl Hepes buffer, Earle’s salts, and
L-glutamine), supplemented with 20% heat inactivated GG-free bovine serum albumin, l mM pyruvate, 100 pg per ml Gentamicin@(M.A. Bioproducts, Walkersville, MD), and 1 unit per ml heparin. The oocytes were placed into sterile 8chamber tissue culture slides (Lab-Tek Products, Napierville, IL) and incubated at
37°C in a moist atmosphere of 5% C02 in air. The cultures were observed every 24
hours with an inverted microscope. The number of oocytes collected and their stage
of maturation were recorded.
Semen Collection and Insemination
Sperm collected by electro-stimulation were evaluated for percent motility,
percent progressive motility, percent immature sperm, and sperm concentration.
The sperm were then held at 37°C for 5-10 minutes. The oocytes were inseminated
with 0.05 ml of sperm suspension 20-21 hours after oocyte recovery (106-106
s p e d m l ) to allow oocyte maturation.
Collection and Preparation of Homologous Serum
Squirrel monkey blood samples were collected to compare the effect of homologous serum with fetal calf serum in TC-199 medium on fertilization and growth
of embryos in vitro. Samples were taken from the femoral vein of female squirrel
monkeys and allowed to clot overnight, after which the serum was collected and
heated to 56°C for 30 minutes before use.
Criteria of Maturation and Fertilization
At intervals of 24 hours, the cultures were examined and the stage of development recorded. An oocyte was considered mature if one polar body was noted.
The criteria for fertilization were: a) two or more polar bodies observed in the
perivitelline space, b) two or more polar bodies and two or more symmetrical
blastomeres 24 hours after insemination, c) observation of the sperm midpiece or
tail within the cytoplasm. An oocyte that possessed one or more of the above
characteristics was considered fertilized.
Polyspermy
A retrospective analysis of control preparations of previous in vitro fertilization trials over the past 15 years was carried out. The incidence of triploidy a t
various sperm concentrations was determined based on the number of pronuclei
within the cell.
40 / Pierce et al.
TABLE I. Follicular Development of Squirrel Monkeys Receiving Induction Reghens
FSH-HCG
(control)
2mgCC
HCG
4mgCC
HCG
6mgCC
HCG
PGE,
HCG
PGE,
alone
No. of monkeys
23
20
20
22
20
20
Mean no. of follicles
Large
Medium
Small
No. of oocytes
No. atretic oocytes
1.1
3.6
8.3
43
3
0.8"
2.7
5.4"
20
(7)
0.7"
2.0
6.0"
8
4b
(50)
(50)
1.0
3.6
8.9
47
13"
(28)
0.7"
2.8
6.7
24
4
(16)
1.4
3.6
5.1"
40
7
(17)
15
(38)
(0)
6
(60)
5"
(15)
16
(80)
18
(55)
1
(25)
2
(50)
9
1
1
(31)
(25)
(7)
1.0
2.0
(%)
No. mature
at recovery
(%)
No. mature
(37 h. post HCG)
t%)
No. oocytes
Der monkev
1
(7)
0"
1.8
~~~
0.4
lob
1.2
2.0
~
"Significantly different from control (P< 0.05).
bSignificantlydifferent from control (P< 0.01).
Culture Medium Analysis
Various properties of the culture medium was analyzed at 0,24, and 48 hours
after addition of oocytes, sperm, and culture in the incubator using oocytes obtained following the different induction regimes. Starting osmolarity ranged from
280 to 300 mOsm and was determined using a freezing point osmometer (Osmette,
Precision Systems, Inc., Natick, MA). Calcium concentration was analyzed using a
CalcetteB (Precision Systems, Natick, MA) at the various time intervals. This
instrument measures total calcium concentration in aqueous solutions using a
fluorometric titration technique. The pH of the medium was determined using a
Model 501 digital ionalyzer (Orion Research Inc., Boston, MA) and a system 1304
pWblood gas analyzer (Instrumentation Laboratory, Lexington, MA). The pCOz,
POz, bicarbonate content, total C02 content, standard bicarbonate, and oxygen
saturation were measured using the system 1304 pWblood gas analyzer.
Statistical Analysis of Data
The Student-Newman-Keuls test and analysis of variance (ANOVA)were used
to test the hypothesis that there was no difference between follicular development
and induction regimens. A chi-square test was used to examine the hypothesis that
fertilization rates were higher in the control group than in the treatment groups.
The relationship between in vitro maturation, fertilization, and culture medium
variables was evaluated using Pearson's linear correlation.
RESULTS
Follicular Induction
The results of follicular development trials and oocyte yield are shown in Table
I. Follicular growth of large and small follicles in monkeys receiving either 2 or 4
mg of clomiphene citrate was significantly lower (P < 0.05) than controls. Animals
Follicular Induction / 41
TABLE 11. Characteristics of Fetal Calf Serum and Homologous Serum
Characteristic
and unit
PH
PCOZ
PO2
HCO, (mmol/L)
TCOze (mmovL)
SBCb
%SOZC"( m m O v L )
Osmolarity (mOsm)
Calcium (mEq/L)
Fetal calf serum
(Mean f SD)
Squirrel monkey serum
(Mean f SD)
7.00 f 0.04
39.9 f 4.31
146 f 4.95
10.1 k 0.07
11.3 f 0.21
9.3 f 0.71
98.0 2 0.57
309 f 1.41
6.98 f 0.23
7.60 .t 0.02
16.1 t 3.56
133 f 5.01
18.8 f 0.09
19.3 f 0.30
20.5 2 0.66
99.6 f 0.19
304 2 0.04
4.30 f 0.28
Total carbon dioxide.
bstandard bicarbonate content.
'Percent oxygen saturation content.
given 6 mg of clomiphene showed growth of follicles similar to those of the control
group. There were significantly fewer large follicles in the prostaglandin Elplus
HCG group and smaller follicles in the prostaglandin El alone group (P < 0.05)
than in the controls. A significantly higher percentage of atretic oocytes was recovered from the low and medium doses of clomiphene (P < 0.01)and the high dose
(P < 0.05) when compared to the controls. Significantly fewer mature ova were
collected from monkeys given 2 or 6 mg of clomiphene citrate (P < 0.05).
Comparison of Serum Source
Homologous serum (20%) pooled from several monkeys was substituted for
fetal calf serum in TC-199culture medium in order to evaluate its effect on in vitro
fertilization and subsequent development. Table I1 shows the culture characteristics of fetal calf serum and homologous serum. There was no significant difference
in fertilization rate (P < 0.05) between serum types, but the number of embryos
cleaving to the two-cell stage was greater (P < 0.05) when fetal calf serum was the
protein source (Table 111).
In Vitro Fertilization Following Follicular Induction
Some oocytes obtained from all of the follicular induction regimens were capable of being fertilized (Table IV)but there were no significant differences among
the treatment and control groups for fertilization rate. No embryos cleaved beyond
the two-cell stage in the 2- or 4-mg clomiphene citrate groups, while development
proceeded to the 4- to 8-cell stage in the remaining groups.
Culture Medium Analysis
In analyzing the culture media, the following characteristics were found The
PC02and POz changed the most throughout all experiments while pH, bicarbonate concentration,TC02, standard bicarbonate content, percent oxygen saturation,
and osmolarity remained stable over the 48-hour culture period. There was a
strong inverse relationship between in vitro maturation rate and calcium concentration (r2 = 0.98).No correlation between maturation or fertilization rates and
the remaining culture characteristics among the follicular induction treatments
was found.
42 / Pierce et al.
TABLE 111. Comparison of Fertilization and Early Development of Squirrel Monkey
Oocytes in Fetal Calf Serum or Homologous Serum in TC-199 Culture Medium
20%Fetal
calf serum
20% Homologous
serum
19
27
3 (11.1%)
5 (21.0%)
8/24 (33.3%)
22
49
6 (12.2%)
10 (23.2%)
11/43 (25.6%)
2%
Factors
No. animals
No. eggs collected
No. atretic eggs
No. mature eggs at recovery
No. fertilized/No. inseminated
No. two-cell embryos.
4
*P < 0.05 significant.
TABLE IV. Fertilizability of Oocytes Recovered From Squirrel Monkeys Receiving
Follicular Induction Regimens*
"rial:
No. of monkeys
No. of oocytes
No. of mature oocytes
No. fertilized
(%)
No. developed beyond
two-cell stage
(%)
No. viable
(i.e., not atretic)
oocytes per monkey
FSH-HCG
(control)
2mgCC
HCG
4mgCC
HCG
6mgCC
HCG
PGE,
HCG
PGE,
alone
23
43
16
14
(87)
20
8
1
1
(100)
20
20
8
6
(75)
22
47
14
14
(100)
20
24
17
11
(65)
20
40
19
14
(74)
2
(14)
(0)
0
(0)
5
(36)
1
(9)
1
(7)
1.7
0.2
0.5
1.5
1.0
1.7
0
*Values were not statistically different from controls.
Polyspermy
A retrospective study of 28 previous control in vitro fertilization experiments
found the incidence of polyspermy to be 11%(Table V). The culture medium used
in all the experiments was TC-199 supplemented with 20% fetal calf serum and pH
ranged from 7.3 to 7.8. A total of 107 oocytes were fertilized (60%) and 12 ova were
found to be polyspermic. Polyspermy occurred over a range of sperm concentrations
(1.4 x 104/ml to 4.7 x 106/ml) and there was no correlation found between the
incidence of polyspermy and sperm concentration.
DISCUSSION
These studies indicate that the squirrel monkey is a suitable model for examining aspects of in vitro fertilization, including follicular induction regimens, culture conditions, and polyspermy. Previously, induction of follicular growth has
been accomplished in the squirrel monkey with pregnant mare's serum [Bennett,
1967; a u l d et al., 19731, follicle-stimulating hormone [Dukelow, 19701, and human urinary follicle-stimulating hormone [Yano & Gould, 19851. Clomiphene citrate has been successfully used to induce follicular growth in the baboon [Irsigler
et al., 19841 and humans [Holman & Hammond, 19881.
This study examined three doses of clomiphene citrate for inducing follicular
Follicular Induction / 43
TABLE V. Incidence of Polyspermy in the Squirrel
Monkey In Vitro Fertilization System
No. of experiments
No. of oocytes
No. of mature oocytes
(16 hours post HCG)
No. of fertilized oocytes
No. of polyspermic oocytes
Average no. s p e d m l used
for fertilization
Average no. s p e d m l
of non-polyspermic oocytes
Average no. s p e d m l of
polyspermic oocytes
28
486
179 (37%)
107 (60%)
12 (11%)
5.1 x 10'
3.4 x 106
8.8
X
10'
growth and oocyte yield in the squirrel monkey. Oocyte production with 6 mg (8
mgkg bwt) of clomiphene citrate for four days showed similar effectiveness as the
standard FSH-HCG regimen. Irsigler et al. [19841used a dose Of 5 mgkg for follicle
induction in baboons and reported collecting an average of five oocytes per animal.
Of those ova, 20% were atretic, 51% were mature, and the rest immature. In
chimpanzees, one oocyte was aspirated per animal after administration of 1.4
mgkg clomiphene citrate for five days [Gould, 19831. The present study examined
doses of 3, 6, and 8 mgkg body weight and 0.4, 1, and 2 oocytes per animal were
collected, respectively. Gould 119831 reported growth of one large follicle in 58%of
the animals and development of one or more follicles in 42%of the cases. This is
similar in the squirrel monkey with an average of one follicle growing per animal,
but there was development of medium and small size follicles in almost all of the
animals.
There were significantly more atretic oocytes collected from animals given
clomiphene citrate. Although the exact cause is not known, Littman and Hodgen
[1985] speculated that elevated luteinizing hormone/HCG levels may increase androgen production by the thecal cells, thereby promoting follicular atresia. It is
unclear whether clomiphene citrate acts on the hypothalamus, pituitary, or the
ovary to cause this early rise in luteinizing hormone which has been observed in
cynomolgus monkeys (M.
fasciculuris).
There were significantly fewer follicles (large and small) with the two lowest
doses of clomiphene citrate. Marrs et al. [19841 reported that clomiphene alone
may not provide enough gonadotropin support for multiple follicle development.
For this reason, Balmaceda et al. [19841 chose to use human menopausal gonadotropin over clomiphene since HMG yielded a larger number of mature oocytes at
aspiration.
There was little difference in follicle growth and oocyte production in the
prostaglandin El treatment groups. In the control group, 38% of the ova were
mature at collection, which is similar to previous reports for this regimen [Kuehl
and Dukelow, 1975al. However, when prostaglandin Eland HCG were administered together, significantly more mature oocytes were aspirated at laparoscopy.
This study reports 80%of the oocytes collected after prostaglandin-HCG treatment
were mature. A previous study in M.rnuluttu [Batta & Brackett, 19781 resulted in
75%of the oocytes retrieved being mature, which is comparable to results reported
here. The authors ascribed this effect to the action of prostaglandin El and HCG for
providing the primary stimulus for the resumption of meiosis resulting in oocyte
44 I Pierce et al.
maturation. This synchronization was observed with the squirrel monkeys when
both PGE, and HCG were administered. While prostaglandin El alone increased
maturation, it was not significantly different from the control.
Some oocytes collected from all of the follicular induction regimens were successfully fertilized. There was no significant difference in fertilization rate between the treatment and control groups. A fertilization rate of 87%was achieved
in the control FSH-HCG group, which is comparable to results described earlier for
the squirrel monkey [Chan et al., 19821. Fertilization rates of oocytes aspirated
after administration of clomiphene citrate were slightly higher than in previous
work [Could, 1983; Irsigler et al., 19841. In the baboon, low fertilization was attributed to difficulty in semen collection and processing as well as culture contamination.
The incidence of triploidy caused by polyspermy is known to be greater in in
vitro fertilization systems compared to fertilization in vivo. The rate of polyspermy
has been shown to depend on a range of experimental variables, e.g., sperm concentration, time of preincubation of spermatozoa, age of eggs, and the genotype of
the gametes [Wolf & Inoue, 1976; Kaleta, 1977; Wolf, 19781. Polyspermypreventing mechanisms in the hamster have been discussed [Barros & Yanagimachi, 19721, and it was suggested that delays in in vitro culture had an adverse effect
or weakened the polyspermy-preventing mechanisms at the zona pellucida and
vitelline membrane, resulting in an abundance of capacitated sperm and multiple
entry.
The effect of sperm concentration [Wolf et al., 19841 and various ovarian stimulation protocols have been examined in several human in vitro fertilization programs [Diamond et al., 1985a,bl. Wolf et al. 119841 reported that maximal fertilization (80.8%) occurred at a concentration of 2.5 x lo4 motile s p e d m l . The
incidence of polyspermy was directly related to sperm concentration increasing
from 0% at 1-2.2 x lo4 to 5.5% at 10 x lo4 motile s p e d m l . An inverse relationship between fertilization and sperm concentration was observed with immature oocytes cultured in vitro and inseminated with a maximal fertilization rate of
66.6%at 5 x lo4 motile s p e d m l . Diamond et al. [1985a,bl examined sperm
concentrations and several ovulation induction protocols with respect to polyspermic fertilization. Polyspermy occurred equally in mature and immature oocytes at
a rate of 10.2%and a significant difference in polyspermy was observed between
stimulating regimens using clomiphene citrate (CC) and those using human menopausal gonadotropin (hMG) or those combining hMG and CC.
A retrospective study was done to examine the incidence of polyspermy and
sperm concentration in our in vitro fertilization system. High sperm concentrations are used in human in vitro fertilization systems [Lopata et al., 1980; Trounson et al., 19811 and it was thought that the large concentration of sperm might
account for this observation. The present study found no correlation between
sperm concentration and polyspermy since ova became polyspermic with concentrations ranging from 1.4 x lo4 to 4.7 x lo6 s p e d m l . The incidence of
polyspermy in the control experiments examined was 11%,which is comparable to
a previous report [Asakawa & Dukelow, 19821. Irsigler et al. [19841 reported
polyspermy in only 2 out of 90 baboon oocytes fertilized in vitro.
This work examined culture medium characteristics over a period of 48 hours
and looked for any relationship to in vitro maturation and fertilization. The basic
medium used in all of the experiments was modified TC-199 (with Earle’s salts)
supplemented with 20%fetal calf serum, sodium pyruvate, gentamicin, and heparin. Initial pH and osmolarity were adjusted to 7.4 to 7.5 and 280-300 mOsm,
respectively. The pH remained stable when incubated in a 5% C0,-moist air at-
Follicular Induction / 45
mosphere. This is in agreement with Bavister [19811,who stated that bicarbonateCO, buffer system yielded the best results for fertilization in hamsters. The pH in
those experiments decreased from 7.6 to 7.5 with TALP medium while in the
current squirrel monkey system, pH generally increased from 7.4 to 7.5 in the
TC-199.The optimal pH for in vitro fertilization in hamsters is 7.4 [Bavister, 19811
with higher pH levels leading to an increased incidence of polyspermy [Bavister,
19691.However, the pH of squirrel monkey serum was 7.67 so a slight increase in
pH from 7.5 to 7.6 would probably not be detrimental. The fact that the pH of the
TC-199 cultured in a bicarbonate-COz system remains stable over a period of at
least 48 hours is encouraging.
The osmolarity of the medium also did not change significantly over time. A
wide range of osmotic pressures have been reported to support in vitro fertilization
[Miyamoto & Chang, 1973;Bae & Foote, 19801. The latter group of researchers
described the highest in vitro maturation of rabbit oocytes a t 270 mOsm, but this
was not significantly different for osmotic pressures ranging from 250-310 mOsm.
No differences were noted in these experiments regardless of the source of
serum (fetal calf serum or homologous squirrel monkey serum) added to the basic
medium.
Oxygen saturation (%SOz) and total carbon dioxide content (TCO,) remained
quite constant throughout time in these experiments. The normal human values
are 96-97% and 23-27 mmoVL [Altman, 19611, respectively, while values for
squirrel monkey serum were comparable at 99.6 f 0.2% and 19.3 f 0.3 mmoVL.
Actual bicarbonate content and standard bicarbonate content (SBC) for squirrel
monkey serum were 18.8 k 0.1 mmol/L and 20.5 k 0.7mmolL, which is slightly
lower than reported in humans (22-26 mmoyL and 22-26 mmoliL, respectively)
[Altman, 19611.These values remained stable over time in the follicular induction
experiments, which aided in stabilizing the pH of the medium.
The pC0, and pOz did vary somewhat throughout the 48-hour period. In all of
the experiments except the 4 mg of clomiphene and prostaglandin Elalone, the
percent of carbon dioxide usually decreased from 5% to 4%. The starting pC0, in
the two aforementioned experiments was low (3-4% and dropped to 1.7 and 2.2%,
respectively). However, it did not have an adverse effect on in vitro maturation or
fertilization. The average starting PO, for all six experiments was 168 mmoVL
(23% oxygen atmosphere) and decreased to 135 mmoVL (19% oxygen) after 48
hours. The oxygen atmosphere in this study is within the acceptable range reported by others [Wright et al., 1976;Peters et al., 19771.Since the embryos utilize
oxygen in the oxidation of pyruvate, oxygen tension would be expected to decrease
[Betterbed & Wright, 19851.
Calcium is known to play a n important role in reproduction (acrosome reaction, sperm binding, oocyte maturation, and cell division). Calcium concentrations
ranged from 3.2 to 4.0 mEqL in the follicular induction experiments. An inverse
relationship was found between in vitro maturation and calcium levels (3= 0.98),
suggesting that high calcium concentrations in culture have an adverse effect on
maturation.
A better understanding of optimal in vivo conditions and more precise culture
techniques should aid in increasing mammalian in vitro fertilization rates in species where in vitro fertilization has been successful, sporadic, or not reported at all.
CONCLUSIONS
The squirrel monkey in vitro fertilization system is useful in studying early
embryo development, culture conditions, and physiological phenomena such as
polyspermy. The following conclusions resulted from the data obtained
46 I Pierce et al.
1. Homologous serum is an adequate protein source in TC-199 fertilization
medium for squirrel monkey oocytes.
2. Induction of follicular growth with 2 and 4 mg of clomiphene citrate was
poor. Clomiphene citrate increased the incidence of atretic follicles at all levels
tested.
3. Injection of animals with prostaglandin Elor prostaglandin Elplus HCG
resulted in more mature oocytes at laparoscopy. The prostaglandin El did not
enhance follicular growth but did synchronize oocyte maturation.
4. Oocytes resulting from the follicular induction regimens were capable of
fertilization and development. The incidence of polyspermy was not dependent on
sperm concentration.
ACKNOWLEDGMENTS
Partially supported by NIH Grants HD07534 and ES04911. Appreciation is
expressed to Mrs. L. M. Cleeves for preparation of the manuscript.
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FLUIDS. D.S. Dittmer, ed. Washington,
DC, Federation of American Society for Experimental Biology, 1961.
Asakawa, T.; Dukelow, W.R. Chromosomal
analyses after in uitro fertilization of squirrel monkey (Sairniri sciureus) oocytes. BIOLOGY OF REPRODUCTION 26579583, 1982.
Bae, I.-H.; Foote, R.H. Maturation of rabbit
follicular oocytes in a defined medium of
varied osmolarity. JOURNAL OF REPRODUCTION AND FERTILITY 59~11-13,
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