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Evaluation of the vervet (Clorocebus aethiops) as a model for the assisted reproductive technologies.

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American Journal of Primatology 69:917–929 (2007)
RESEARCH ARTICLE
Evaluation of the Vervet (Clorocebus aethiops) as a Model
for the Assisted Reproductive Technologies
MICHELLE L. SPARMAN1, CATHY M. RAMSEY1, CARRIE M. THOMAS1
SHOUKHRAT M. MITALIPOV1, JOHN W. FANTON2, GWEN M. MAGINNIS2
RICHARD L. STOUFFER1,3, AND DON P. WOLF1
1
Division of Reproductive Sciences, Oregon National Primate Research Center,
Beaverton, Oregon
2
Division of Animal Resources, Oregon National Primate Research Center,
Beaverton, Oregon
3
Department of Obstetrics and Gynecology, Oregon Health & Science University,
Portland, Oregon
The vervet monkey was evaluated as a primate model for use in assisted
reproductive technologies (ARTs). Eight adult female vervets were
hormonally monitored for their potential use as egg donors and those
six females displaying regular menstrual cycles were subjected to
controlled ovarian stimulation with recombinant human gonadotropins.
Three animals failed to respond while laparoscopic follicular aspiration
was performed on the other three females at 27–30 h post-human
chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes
was recovered from these three animals of which 30, 20, and 4,
respectively, matured to the metaphase II stage and were subsequently
inseminated using intracytoplasmic sperm injection. An average of
40715% (SEM) of the inseminated oocytes were fertilized based on
pronucleus formation and timely cleavage. One embryo from each of the
two stimulated females developed into expanded blastocysts. Two adult
male vervets were assessed as sperm donors. Neither adjusted well to the
restraint and collection procedure required for penile electroejaculation.
Samples collected via rectal electroejaculation were very low in sperm
motility and concentration; however, cauda epididymal aspirations
from one male yielded an adequate concentration of motile sperm. These
results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be
considered for basic and applied (ARTs) research on primate gametes or
embryos. Am. J. Primatol. 69:917–929, 2007. c 2007 Wiley-Liss, Inc.
Key words: vervet; ovarian stimulation; embryo
Contract grant sponsor: Oregon National Primate Research Center (ONPRC); Contract grant
number: RR00163.
Correspondence to: Don P. Wolf, Division of Reproductive Sciences, Oregon National Primate
Research Center, Oregon Health & Sciences University, West Campus, 505 NW 185th Avenue,
Beaverton, OR 97006, USA. E-mail: wolfd@ohsu.edu
Received 27 April 2006; revised 24 October 2006; revision accepted 7 November 2006
DOI 10.1002/ajp.20413
Published online 14 March 2007 in Wiley InterScience (www.interscience.wiley.com).
r 2007 Wiley-Liss, Inc.
918 / Sparman et al.
INTRODUCTION
The vervet (Clorocebus aethiops) has the potential to be a valuable research
model for studying the assisted reproductive technologies (ARTs). Unlike the
commonly studied rhesus macaques, vervet females are believed to maintain
a menstrual cycle year-round and have a straight cervix. Vervet males have been
reported to possess human-like sperm characteristics (e.g. concentration,
motility, pH of seminal fluid, acrosomal integrity) [Hess et al., 1979; Van Der
Horst et al., 1999]. The rhesus macaque has been used extensively as a model for
early human development and for fertilization and embryonic development in
primates. Over the past 20 years, remarkable advancements in the ARTs have
been achieved using the rhesus monkey model. Several of these accomplishments,
including multiple follicle stimulation, oocyte fertilization by intracytoplasmic
sperm injection (ICSI), and oocyte/embryo manipulation, were recently described
[Wolf, 2004]. For a particular species such as the vervet to serve as an alternative
model for studying the ARTs, the ability to obtain a high number of healthy,
fertilizable oocytes, collect and cryopreserve fertile spermatozoa, as well as
cryopreserve and transfer the resulting embryos when a recipient is available
would be essential.
Initial attempts to stimulate ovaries of vervet females required repeated
administrations of equine and hCG [Sankai et al., 1997]. In these studies,
conventional in vitro insemination of meiotically mature, vervet oocytes led to a
high incidence of polyspermy. We report on the recovery of mature oocytes
following controlled ovarian stimulation (COS) of vervets with recombinant
human gonadotropins, fertilization by ICSI, and subsequent embryonic development to the blastocyst stage using assisted reproductive techniques developed for
rhesus macaques.
MATERIALS AND METHODS
Animals
Two adult vervet males, weighing 7.0 and 8.8 kg, and eight adult vervet
females, weighing 3.1–4.9 kg, were studied at the Oregon National Primate
Research Center (ONPRC). All animals were acquired from the Behavioural
Science Foundation at Basseterre, St. Kitts, Eastern Caribbean. The general care
and housing of vervet monkeys by the Division of Animal Resources, ONPRC, was
described previously [Molskness et al., 2007]. Animal protocols in this study were
reviewed and approved by the ONPRC IACUC.
Semen Collection and Cryopreservation
Fresh semen was collected from two adult male vervet monkeys. The fertility
of both males was unknown before this study. Efforts to harvest progressively
motile sperm involved the use of penile electroejaculation [Lanzendorf et al.,
1990], rectal electroejaculation, and cauda epidydimal aspiration on anesthetized
males. It should be noted that the epidydimal aspiration technique was performed
only as a final attempt to recover a viable sperm sample from each male for our
fertilization efforts. Electroejaculated semen samples were allowed to liquefy for
15–30 min before the liquefaction of the coagulated ejaculate was removed
[Mitalipov et al., 2002]. Aspirated epidydimal tissue was immediately placed
into Hepes-buffered Talp (modified Tyrode solution with albumin, lactate, and
pyruvate) medium [Bavister et al., 1983] containing 0.3% bovine serum albumin
(TH3) and transported to the laboratory for processing. All semen samples were
Am. J. Primatol. DOI 10.1002/ajp
The Vervet as a Model for the ARTs / 919
washed two times in TH3 medium by centrifugation at 1,400 RPM for 8 min
[Sanchez-Partida et al., 2000]. Sperm motility and concentration were examined
in each sample. Sperm samples were either used immediately for in vitro
fertilization of oocytes or cryopreserved and stored in liquid nitrogen.
For cryopreservation, the washed sperm pellet was resuspended in a TESTris buffer solution containing 30% egg yolk, 20% skim milk, 3% glycerol, and
0.06 M glucose and cooled to 41C for 90 min [Tollner et al., 1990]. This sperm
suspension was deposited as 20–50 ml drops into depressions on the surface of dry
ice. The frozen drops were transferred into cryovials and stored on canes in liquid
nitrogen. For thawing, a sperm pellet was transferred into a dry test tube and
together were partially submerged into a 371C water bath (o1 min). The thawed
sample was washed in TH3 medium and evaluated as described above.
Detection of Menstruation and Hormone Measurement
Females were vaginally swabbed on a daily basis to detect the onset of
menstruation. The first day of menstruation was considered day 1 of the
menstrual cycle. On a weekly basis, blood samples were collected from each
female and analyzed for serum hormone levels by the Endocrine Services Core
Laboratory, ONPRC. Serum estradiol (E2) and progesterone (P4) levels were
measured using electrochemoluminescent assays [Young & Stouffer, 2004] and a
Roche Elecsys 2010 analyzer (Roche Diagnostics, Indianapolis, IN). The detection
of menses and the serum hormone levels were used to determine the regularity of
monthly menstrual cycles [Eley et al., 1989; Molskness et al., 2007]. Serum
luteinizing hormone (LH) levels were also measured by the Endocrine Services
Core Laboratory in samples from two females during ovarian stimulation
protocols when a spontaneous LH surge was suspected. The Core personnel
stimulated mouse interstitial cell production of testosterone in vitro to determine
serum LH [Yeoman et al., 1988].
Stimulation of Ovaries
Multiple follicular development in the ovaries was induced in females,
exhibiting regular menstrual cycles, using exogenous gonadotropins. A COS
protocol, designed and used previously in rhesus macaques to generate numerous
mature oocytes for in vitro fertilization, was employed in this study [Meng &
Wolf, 1997; Wolf et al., 1990, 2004; Zelinski-Wooten et al., 1995]. Starting at
menses (days 1–4 of the menstrual cycle), females received daily injections of a
GnRH antagonist (Antide; Ares Serono, Aubonne, Switzerland; 0.5 mg/kg body
weight, subcutaneous) to prevent an endogenous LH surge, plus recombinant
human follicle stimulating hormone (r-hFSH; Gonal F; Ares Serono; 30 IU,
intramuscular, twice daily) for 8 days. Recombinant human LH (r-hLH; Ares
Serono; 30 IU, intramuscular, twice daily) was administered along with r-hFSH
and GnRH antagonist on days 7 and 8 of the stimulation. All other reagents used
in this study were from Sigma-Aldrich Co. (St. Louis, MO) unless otherwise
indicated. Ultrasonography was performed starting on day 7 to assess ovarian
response. Females with enlarged ovarian follicles 3–4 mm in diameter received
hCG (Serono, Randolph, MA; 1,000 IU, intramuscular) to induce the resumption
of oocyte maturation [Zelinski-Wooten et al., 1994]. Blood samples were collected
daily during the stimulation and analyzed for E2 and P4 levels, as described
earlier.
Am. J. Primatol. DOI 10.1002/ajp
920 / Sparman et al.
Oocyte Collection and In Vitro Fertilization
Oocyte collection followed previously described protocols for the rhesus
macaque [Wolf et al., 2004; Zelinski-Wooten et al., 1995]. Laparoscopic follicular
aspiration was performed on anesthetized monkeys 27–30 h post-hCG injection
[Wolf et al., 1996] by the Department of Surgery, Division of Animal Resources,
ONPRC. Tubes containing follicular aspirates were transported to the laboratory
in a portable incubator (Minitube, Verona, WI) at 371C. The aspirates were sifted
through a cell strainer (Becton-Dickonson, Franklin Lakes, NJ; Falcon, 70 um
pore size) and then immediately backwashed with Hepes-buffered Talp (modified
Tyrode solution with albumin, lactate, and pyruvate) medium [Bavister et al.,
1983] containing 0.3% bovine serum albumin (TH3) to recover the oocytes.
Oocytes were stripped of cumulus cells by mechanical pipetting and a brief (30 s)
exposure to hyaluronidase (0.5 mg/ml), examined for determination of developmental stage (germinal vesicle-intact: GV; metaphase I: MI; metaphase II: MII;
degenerate), morphology, color of the cytoplasm, granularity, and then cultured
in HECM-9 medium [McKiernan & Bavister, 2000; VandeVoort et al., 2003; Wolf
et al., 2004] overlaid with paraffin oil (Ovoil, Zander IVF [in vitro fertilization],
Vero Beach, FL) at 371C in 5% CO2 until fertilization. Oocyte maturity
(progression from MI- to MII-stage) was evaluated and recorded approximately
every 2 h following collection until fertilization.
On the day of collection (day 0; d0), meiotically mature MII oocytes were
fertilized with a single vervet spermatozoan using ICSI [Meng & Wolf, 1997;
Mitalipov et al., 2001; Nusser et al., 2001]. Injections were performed at 371C in
30 ml drops of TH3 medium in the lid of a 60 mm tissue culture dish, covered with
paraffin oil, using an Olympus inverted microscope (Melville, New York, USA)
equipped with micromanipulators (Narishige, Tokyo, Japan). Sperms were placed
in a 4 ml drop of 10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA),
immobilized, aspirated tail first into a microinjection needle (Humagen,
Charlottesville, VA), and injected into the cytoplasm of each oocyte. Following
ICSI, all oocytes were returned to a 4-well dish (Nalge Nunc International Co.,
Naperville, IL) containing HECM-9 and cultured overnight at 371C in 6% CO2, 5%
O2, balance N2.
Embryo Culture and Cryopreservation
Successful fertilization, determined by the presence of two pronuclei, was
assessed 14–16 h post-injection. Cleavage-stage embryos were cultured in 4-well
dishes in HECM-9 medium containing 5% fetal bovine serum (Hyclone, Logan,
UT) overlaid with paraffin oil at 371C in 6% CO2, 5% O2, balance N2. The
progression of embryonic growth was assessed daily and culture medium was
changed every other day. Embryos were either cultured under these conditions
to the blastocyst stage or cryopreserved on day 3 for future thaw and transfer into
a surrogate female. Embryos designated for cryopreservation were placed in
increasing concentrations of the cryoprotectant propanediol [Wolf et al., 1989],
transferred to a cryovial, and cooled at a rate of –21C/min to a seeding
temperature of 71C in a BioCool control rate freezer (FTS Systems, New York,
USA). Once ice crystal formation was manually initiated, the freezer temperature
was dropped at 0.31C/min to 301C. The cryovial containing embryos was then
plunged into liquid nitrogen for storage. Thawing was performed initially at 371C
followed by the dilution of the propanediol in a step-wise manner at room
temperature [Wolf et al., 1989].
Am. J. Primatol. DOI 10.1002/ajp
The Vervet as a Model for the ARTs / 921
Embryo Transfer
Embryo transfer procedures were similar to those described for rhesus
macaques [Nusser et al., 2001; Wolf et al., 2004]. Blood samples were collected
daily starting on day 8 of the menstrual cycle and E2 levels were determined by
radioimmunoassay. The day following the E2 peak is commonly referred to as day
0 [Wolf et al., 2004]. This information was used to determine the timing of
the embryo transfer. On day 1, 2 days after the estradiol peak the embryos were
thawed and placed in culture the afternoon preceeding the scheduled day of
transfer. On day 2, embryo survival was assessed based on the following criteria:
presence of the zona pellucida, recovery of 450% of the blastomeres, and overall
embryo morphology and quality [Wolf et al., 2004]. Embryos that survived were
placed in TH3 medium and immediately transported to the operating room at
ONPRC. A surgical embryo transfer [Nusser et al., 2001; Wolf et al., 2004] was
performed, with the assistance of the Department of Surgery at ONPRC, by
transferring two frozen–thawed vervet embryos into an oviduct of an anesthetized recipient vervet female (3–4-day old embryos into a day 2 oviduct). The
embryos were deposited approximately 2 cm into the oviduct via the fimbria on
the side with an ovulation site. Blood samples were collected daily and analyzed
for serum E2 and P4 levels and ultrasonography was performed on day 35 posttransfer to determine implantation and pregnancy.
RESULTS
Response to Controlled Ovarian Stimulation
Eight females were screened as potential egg donors, with six maintaining
regular menstrual cycles. Multiple follicular development was successfully
induced in three of six regularly cycling monkeys, with exogenous gonadotropins
as determined by ultrasonography (Fig. 1a and b; female 1). Figure 2a and b
display the E2 and P4 levels, respectively, measured during the COS cycle. Three
females that responded to the stimulation (successful COS) had E2 levels
exceeding 4,000 pg/ml between days 10 and 12 of the menstrual cycle. A peak E2
level of 6,27071,223 pg/ml (mean7SEM) was detected on day 11, whereas a
P4 peak of 5277 ng/ml (mean7SEM) was measured on day 18. These peak
estradiol and P4 levels were 4ten-fold higher than levels observed in the two
females displaying what seemed like an abbreviated response (E2: 568 pg/ml; P4:
4.3 ng/ml; mean). This abbreviated-like response was not associated with a
spontaneous LH surge during hormonal treatment (based on LH bioassay
results). Additionally, one female did not respond to the exogenous r-hFSH
treatment and the protocol was terminated on day 8 of treatment. Menstruation
was detected in all three successful responders 17–20 days post-hCG.
Spermatozoa Motility and Concentration
Table I depicts the results of semen analysis on samples recovered by various
collection methods from two male vervet monkeys. Multiple attempts to collect
semen samples by penile electroejaculation were not successful as neither male
adjusted to the restraint apparatus and collection procedures. Most samples
obtained by rectal electroejaculation had few or no motile spermatozoa, except
one ejaculate that yielded approximately 0.2 106 sperms/ml with 28% motility.
In a final attempt to recover spermatozoa for in vitro fertilization, a biopsy of
epidydimal tissue was surgically taken from each male. An adequate concentration of motile spermatozoa was obtained from the epidydimal tissue of one of the
Am. J. Primatol. DOI 10.1002/ajp
922 / Sparman et al.
A.
B.
Fig. 1. Ultrasonographic images depicting large multiple follicles in ovaries of one successful
controlled ovarian stimulation animal (female 1; day 9 of treatment). (A) Left ovary is depicted by
LO in the top image. (B) Right ovary is depicted by RO in the lower image; both from the same
vervet female. Numerical values on the ovary represent the diameter of each follicle (in
millimeters). The numerical value next to the RO and LO text represents the measured diameter
of each ovary (in millimeter).
two males. The two samples with measurable sperm populations were used for
ICSI (both samples) and to evaluate sperm survival post-thaw (only one sample).
Inadequate motility (low percentage: 32%; minimal progressive movement,
Am. J. Primatol. DOI 10.1002/ajp
The Vervet as a Model for the ARTs / 923
8,000
Hormonal Stimulation
hCG
Serum Estradiol Level (pg/ml)
7,000
6,000
5,000
Successful COS (n=3)
Abbreviated COS (n=2)
No response (n=1)
4,000
3,000
2,000
1,000
0
5
Serum Progesterone Level (ng/ml)
A.
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Day of Menstrual Cycle
70
hCG
60
50
Successful COS (n=3)
Abbreviated COS (n=2)
No response (n=1)
40
30
20
10
M
0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
B.
Day of Menstrual Cycle
Fig. 2. (A) Serum estradiol (E2) levels measured during controlled ovarian stimulation (COS) cycles
of six female vervets treated with human gonadotropins to stimulate multiple follicular
development. The bar at the top of the figure depicts the interval when r-hFSH, r-hLH, and
Antide were administered. The adjacent ‘‘human chorionic gonadotropin (hCG)’’ depicts the timing
of the hCG injection to induce ovulatory maturation. The serum estradiol values for the successful
COS group are the mean of three monkeys that responded positively, with increasing E2 levels
during hormonal stimulation, the abbreviated COS group are the mean of two monkeys that had
lower E2 levels that declined prior to the end of hormonal treatment, and the no response is for the
one monkey that was cancelled early since E2 levels did not increase, in response to the hormonal
treatment. Results expressed as mean7SEM for the successful COS group. (B) Serum progesterone
(P4) levels measured during the presumptive luteal phase in COS cycles of six female vervets
treated with human gonadotropins to stimulate multiple follicular development. The ‘‘hCG’’ and
arrow depicts timing of the hCG injection to induce ovulatory maturation of follicles. The letter ‘‘M’’
and arrow depicts the time period when the onset of menstruation was detected for the successful
COS group (as defined in Fig. 2 legend). The serum progesterone values for the successful COS
group are the mean7SEM of three monkeys, abbreviated COS group are the mean of two monkeys,
and the no response is for the one monkey that was cancelled early during the hormonal treatment.
twitching) and abnormal morphology (bent tails: 42%; broken tails: 39%) of
frozen/thawed spermatozoa were observed.
In vitro Fertilization, Embryonic Development, Embryo Transfer
Table II summarizes the results of oocyte collection, fertilization by ICSI, and
subsequent embryonic development. Meiotically active (MI, MII) oocytes were
recovered from large (3–4 mm diameter) follicles of all three vervets successfully
Am. J. Primatol. DOI 10.1002/ajp
924 / Sparman et al.
TABLE I. Analysis of Vervet Spermatozoa Recovered by Various Semen
Collection Techniques: Penile Electroejaculation, Rectal Electroejaculation,
or Epidydimal Aspiration
Male no.
1
2
Sperm collection technique Total sperm count (million/ml)
Penile electroejaculation
Rectal Electroejaculation
Cauda epidydimal aspiration
Penile electroejaculation
Rectal electroejaculation
Cauda epidydimal aspiration
Sperm motility
No sperm recovered
Too few to accurately assess
230.7 106/ml
No sperm recovered
0.2 106/ml
No sperm recovered
NA
NA
32%
NA
28%
NA
Sperm count, displayed as total million sperm (included non-motile sperm) per ml of TH3 media, and sperm
motility expressed as total percent motile, were determined immediately following the recovery of the sperm
sample. Note that the use of penile electroejaculation, commonly used in rhesus macaques, did not produce a
sperm sample in both males. NA, not assessed, because of the lack of sperm recovered.
TABLE II. Summary of Vervet Oocyte Recovery, Fertilization, and Subsequent
Development to the Blastocyst Stage In Vitro
Oocytes:
No. (%)
Female Total no. Oocytes: Oocytes: Oocytes: Oocytes: MII day 1a No. (%)
(total) Fertilized Blastocysts
no.
oocytes
degen
GV
MI
MII day 0a
1
2
3
62
40
18
20
4
5
7
10
9
30
22
3
21
23
1
32
23
4
18 (60%)
2 (10%)
2 (50%)
1 (9%)
0 (0%)
1 (50%)
Oocytes were recovered from three females (day 0) and assessed immediately for total number and developmental
stages. Degen, degenerated; GV, germinal vesicle intact; MI, metaphase I; MII, Metaphase II. Oocyte maturation
from MI to MII stage was monitored on days 0 and 1. MII stage oocytes were inseminated by intracytoplasmic
sperm injection (ICSI) on days 1 and 2, and fertilization status (based on the presence of two pronuclei within
14–16 h of ICSI) and subsequent embryonic development to the blastocyst stage were recorded every day. The
number fertilized is also expressed as a percent of the MII oocytes that had two pronuclei following ICSI. The
number of blastocysts is also expressed as the percent of fertilized embryos that developed to the blastocyst stage.
Note that seven of the 18 embryos from female 1 were cryopreserved and not used to evaluate progression to the
blastocyst stage.
a
Indicates the day of in vitro culture. d0, day of oocyte recovery.
stimulated with r-hFSH, r-hLH, Antide, and hCG. A total of 46% of the oocytes
from two of the three females (1 and 2) matured to the metaphase II stage of
meiosis on the day of aspiration (d0) and 55% within 24 h of oocyte collection (d1).
An average rate of 55% fertilization by ICSI (Fig. 3a) was achieved in all injected
oocytes (d0 and d1) from two females (1 and 3) as evident by the presence of two
pronuclei within 14–16 h of ICSI (Fig. 3b). In female 1, 80% (13/21) of oocytes
matured at d0 were fertilized, whereas fertilization was 55% (5/9) with oocytes
matured at d1. The poor fertilization rate (10%) in female 2 was likely attributed
to the inability to collect a fresh sperm sample from either male on the day of
oocyte retrieval and the use of frozen-thawed sperm. Total loss of sperm viability
was observed after 3 h post-thaw. A delayed rate of cleavage was detected as early
as day 3 when the embryos were at the 4–6 cell stage and a high rate of
developmental arrest (80%) occurred at the 6–8 cell stage (days 3–4) in
subsequent vervet embryos. One embryo from each of two females developed to
the morula stage on day 6, compact morula stage on day 7, and blastocyst stage
between days 9–11 of culture (Fig. 3c). Only one of the two blastocysts hatched
from the zona pellucida by day 12 of culture. On day 3, the highest scoring
Am. J. Primatol. DOI 10.1002/ajp
The Vervet as a Model for the ARTs / 925
A.
B.
C.
Fig. 3. (A) Micrograph of a metaphase II-stage vervet oocyte during intracytoplasmic sperm
injection (ICSI). Note the presence of the polar body at the 6 o’clock position and the sperm located
in the ICSI pipette on the right side of the image. (B) Micrograph of a fertilized vervet oocyte
containing two pronuclei. Fertilization status was assessed 14–16 h following intracytoplasmic
sperm injection (ICSI) and was based on the presence of two pronuclei in the cytoplasm. (C)
Micrograph of a vervet blastocyst. This embryo was derived from a vervet oocyte fertilized by ICSI
and developed to the blastocyst stage after extended culture in HECM-9 media containing 5% fetal
bovine serum.
Am. J. Primatol. DOI 10.1002/ajp
926 / Sparman et al.
embryos (determined by overall morphology; n 5 7) from one female were
cryopreserved and were not used to assess embryonic development to the
blastocyst stage. Upon thawing, three of the seven cryopreserved embryos
survived, but only two, based on morphological evaluation, were transferred into
a recipient vervet monkey. No pregnancy was established.
DISCUSSION
In this report, a sequential treatment of recombinant human gonadotropins
(r-hFSH, r-hLH, Antide, and hCG) was administered to induce controlled
follicular stimulation in vervet females at doses commonly used for rhesus
macaques [Wolf et al., 1990, 2004]. The only reported COS protocol in vervets
employed equine chorionic gonadotropin (CG) for follicular development and
human CG for ovulatory events [Sankai et al., 1997]. FSH is considered the
predominant gonadotropin needed for follicular growth and development. When
FSH is administered along with LH on days 7 and 8 of the stimulation in rhesus
macaques, this combination results in large preovulatory follicles, 3–4 mm in
diameter [Wolf et al., 1989]. After an injection of hCG for ovulation induction,
a cohort of oocytes can be recovered suitable for fertilization and embryo
production. Non-primate gonadotropin preparations, such as eCG, are available
however use of these hormones for macaques results in the production of
antibodies even after one COS cycle [Bavister et al., 1986]. The use of
recombinant human gonadotropins (r-hFSH, r-hLH, and r-hCG) apparently
delays the production of antibodies allowing at least three COS cycles per animal
[Stouffer & Zelinski-Wooten, 2004]. Our ability to recover mature oocytes from
vervet monkeys using the follicular stimulation protocol established for rhesus
monkeys is encouraging. In 2001, Nusser et al. reported that an average of
30 oocytes was recovered per stimulated rhesus monkey. In this study, two of the
three vervet females (1 and 2) provided 40 or more oocytes per animal with over
50% of the total number of oocytes matured to the MII stage of meiosis.
Despite our ability to recover oocytes from three vervet females, two of the six
females displayed what initially seemed like abbreviated responses to the
sequential gonadotropin treatment and one female failed to respond at all to
the exogenous gonadotropins. Since we did not detect an LH surge in the two
females with abbreviated-like responses, it is unlikely that the GnRH-antagonist
(Antide) failed to suppress the animal’s endogenous hormones. This suggests that
three of the six females with regular menstrual cycles failed to fully respond to
the hormonal treatment. It is noteworthy to add that the two abbreviated-like
response females started receiving r-hFSH on day 4 of their menstrual cycle. In
rhesus macaques, we have reduced our stimulation cancellation rate by initiating
all stimulations on days 1–3 of the cycle (unpublished result). A typical
cancellation rate for rhesus macaque stimulations is 22% [Wolf et al., 2004].
To our knowledge, this study is the first to report successful ICSI with vervet
spermatozoa and oocytes. In rhesus monkeys, this insemination technique
eliminates the need to chemically capacitate sperm, minimizes the chance of
polyspermy since only one sperm is injected, and typically results in a high level
of oocyte fertilization and subsequent live offspring following transfer of ICSIderived embryos [Hewitson et al., 1998, 1999; Meng & Wolf, 1997; Nusser et al.,
2001]. Sankai et al. [1997] reported on the first successful in vitro fertilization
(66%) of C. aethiops oocytes using hyperactivated spermatozoa. Of the fertilized
oocytes, over one-third (37%) were polyspermic. Recently, Wolf et al. [2004]
achieved over 80% fertilization rates following ICSI of rhesus oocytes with fresh
Am. J. Primatol. DOI 10.1002/ajp
The Vervet as a Model for the ARTs / 927
or frozen-thawed spermatozoa. We used ICSI in this study because of its success
in the rhesus macaque and the lack of fresh semen samples with adequate sperm
concentration and motility, needed for IVF. Although a 50–60% fertilization rate
of MII oocytes from two vervets is below 80%, these initial ICSI attempts are
encouraging.
In vitro derived rhesus monkey embryos fertilized by ICSI and cultured in
HECM-9 develop to the compacting morula stage on day 3 of culture, blastocyst
stage around day 5, and hatched blastocyst stage as early as day 8 [Wolf et al.,
2004]. These in vitro rates of development are thought to be similar to in vivo
rates of embryonic development [Wolf, 2004]. In comparison, cleavage and
development rates in vitro to the blastocyst stage for vervet embryos were slower.
Sankai et al. [1997] also reported delayed development among in vitro-derived
vervet embryos (morula stage: day 51post-insemination; early blastocyst stage:
day 7; expanded blastocyst stage: day 81). Although it is difficult to pinpoint the
cause(s), it is possible that delayed vervet embryonic development, compared with
rhesus embryos, is a result of sub-optimal culture conditions and/or sublethal
damage to the sperm during processing, cryopreservation, or insemination. The
limited selection of motile vervet spermatozoa for ICSI may also have adversely
affected the developmental competence of subsequent embryos. The slow
progression to the blastocyst stage emphasizes the need to further understand
embryonic development in this species and optimize culture conditions.
Considering that post-thaw survival of rhesus embryos averages 66% (unpublished result), our initial success with the post-thaw survival of vervet embryos
(43%) also supports a promising future for use of the vervet in the ARTs.
The ability to use the vervet in the ARTs was limited by our difficulty in
recovering ejaculated sperm samples. In the rhesus electroejaculation program at
ONPRC if a male proves uncooperative or an ejaculate is not obtained after
several attempts, the animal may be released from the program. Further selection
criterion requires that the males produce ejaculates with over 100 106 sperm
per ejaculate of which over 70% are motile and morphologically normal [Wolf
et al., 1990]. At the California National Primate Research Center (CNPRC), three
males are typically trained to the electroejaculation procedure and the male with
the best semen parameters is selected [VandeVoort et al., 2004]. Unfortunately
we did not have more than two males to evaluate. The semen collection
procedures performed on the vervets are the described techniques for singlecaged rhesus males that regularly provide quality spermatozoa for in vitro
fertilization and cryopreservation efforts [Wolf et al., 1990]. Application of these
procedures to the vervet did not yield the progressively motile spermatozoa
(184 106/ml; 55.5% motility) recovered at other facilities [Seier et al., 1989].
Testing of additional males and species-specific semen collection protocols [Seier
et al., 1989] may help generate ejaculates with normal semen characteristics.
Although sperm motility and concentration from both males were inadequate for
traditional in vitro fertilization, the use of ICSI overcame this limitation. Even
frozen-thawed sperm, nearly immotile and morphologically abnormal post-thaw,
could be used to produce viable embryos. Mdhluli et al. [2004] recently found
similarities in sperm parameters (e.g. concentration, motility, acrosomal
integrity) between vervet and man. These findings further support the potential
value of the vervet as a model for human reproductive research.
With an increasing number of infertile patients using the ARTs, specifically
ICSI, there is a greater focus now on optimizing these techniques to ensure
patient safety. Today it is estimated that over 50% of all IVF cycles in women
involve ICSI [Hewitson, 2004]. This fertilization technique has been associated
Am. J. Primatol. DOI 10.1002/ajp
928 / Sparman et al.
with an increased frequency of specific chromosomal abnormalities and imprinting defects [Bonduelle et al., 1996; Cox et al., 2002]. Non-human primates,
including rhesus macaques and vervets, reach sexual maturity and produce
offspring within a few years making them an ideal model for studying the longterm effects of ICSI and other ART procedures. Here, we show that vervet oocytes
can be collected in large numbers following ovarian stimulation with exogenous
human gonadotropins and fertilized by ICSI using procedures effective in
the rhesus macaque. These results suggest that while modifications to these
procedures are necessary for application in the vervet, the vervet monkey can be
used as a potential model for studying the clinical and scientific application of the
ARTs.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to the surgical staff
at ONPRC for assistance with laparoscopic oocyte retrievals, semen collections,
and the embryo transfer; Division of Animal Resources at ONPRC for hormone
administration, blood draws, and semen collections; and Dr. David Hess and the
Endocrine Services Core at ONPRC for hormone assays. The Serono Reproductive Biology Institute, a member of Serono International, generously donated the
human recombinant hormones and Antide used in this study.
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