| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Primate Biology, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China
b The China-U.S. Primate Biology Laboratory, Kunming Institute of Zoology and University of Wisconsin-Madison, Madison, Wisconsin 53706
c Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
ABSTRACT
Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined. Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and subjected to in vitro fertilization followed by embryo culture. After fertilization, ova from unstimulated prepubertal monkeys displayed lower development to morula (4%) than those from unstimulated adult females (18% in breeding season and 22% in nonbreeding season). No developmental difference was found between ova from unstimulated adult monkeys in breeding and nonbreeding seasons. However, ova from FSH-primed prepubertal monkeys displayed greater development to blastocyst stage (54%) than those from adult monkeys in the breeding season (16%) and nonbreeding season (0%); and ova from FSH-primed adult females in the breeding season had significantly (P < 0.05) greater developmental competence than those obtained in the nonbreeding season (
morula stage, 54% vs. 3%; blastocyst stage, 16% vs. 0%). These data indicate that 1) rhesus monkey oocytes acquire developmental competence in a donor age-dependent manner, and 2) animal age and breeding season modulate the effect of FSH on oocyte developmental competence in the rhesus monkey.
aging, developmental biology, follicular development, FSH, gametogenesis, implantation/early development, IVF/ART, oocyte development, ovum
INTRODUCTION
Mammalian oocytes naturally acquire their competence for maturation, fertilization, and normal development after puberty. In recent years, there has been considerable research on in vitro maturation (IVM), both in order to understand the biology of oocyte maturation and to promote full developmental competence in vitro of immature oocytes from prepubertal and adult animals. IVM of oocytes from adult females has been widely used for studying oocyte maturation and for production of embryos in various species, including mouse [1], cattle [2, 3], sheep [4, 5], pig [6, 7], rhesus monkey [8], and human [9]. However, the in vivo dynamics of acquisition of oocyte competence in prepubertal and peripubertal animals are of particular interest because of the large reservoir of immature oocytes potentially available for embryo production. Studies in cattle and mice have revealed the gradual, age-dependent development of oocyte competence [10, 11].
Mammalian follicle development and oocyte maturation are complex processes in which gonadotropins are the primary regulators [12–14]. Gonadotropins directly control ovarian follicular development via receptors on the follicle cell membranes. In addition, under the action of gonadotropins, follicle cells secrete a range of compounds, including steroids, peptides, and protein growth factors. These locally produced compounds constitute a complex intraovarian paracrine, autocrine, juxtacrine, and intracrine regulatory system that modulates the responses of follicular cells and oocytes to gonadotropic stimuli [15, 16]. Previous studies have shown that the regulatory effect of gonadotropins on oocyte maturation and, more importantly, on the oocyte's capability to sustain embryonic development, is mediated by intraovarian factors [14, 15]. Thus it is proposed that different intraovarian environments will lead to varying responses of oocytes to similar gonadotropin stimulation. For example, in cattle and mice, the ability of FSH to increase the developmental capacity of oocytes matured in vivo or in vitro depends on the age of the oocyte donor [10, 11].
We have found that small antral follicles 1 mm in diameter were already visible not only in the ovaries of adult females during the breeding and nonbreeding seasons, but also in ovaries of prepubertal (1- to 2-yr-old) females. The developmental competence of oocytes and the positive response of oocyte developmental competence to FSH priming have been well established in adult rhesus monkeys during the breeding season [8, 14]. However, little is known about IVM of oocytes collected from prepubertal females or adult females, or the effect of FSH priming on oocytes from prepubertal and adult rhesus monkeys, during the nonbreeding season. The present study was designed to determine 1) if oocytes from prepubertal and adult rhesus monkeys during the breeding and nonbreeding seasons differ in their meiotic and developmental competence, and 2) whether in vivo FSH priming differentially affects the developmental competence of oocytes from prepubertal and from adult monkeys during the breeding and nonbreeding seasons.
MATERIALS AND METHODS
Animals, Treatments, and Oocyte Recovery
Experiment 1. Oocytes from Unstimulated Rhesus Monkeys
All ovaries were excised from unstimulated rhesus monkeys (Macaca mulatta) that were housed outdoors and killed to provide primary cell cultures for a vaccine production program. These animals had never been used for any experiment before they were killed. The adult monkeys (4–15 yr of age) had normal menstrual cycles, but their precise cycle stages at the time of death were unknown. The ovaries were collected into TALP-HEPES medium [17] and transferred to the laboratory within 1–2 h after death. During the breeding season (September to February in Kunming, China), 8 pairs of adult ovaries without apparent corpora lutea and 15 pairs of ovaries from prepubertal monkeys (1–2 yr of age) were collected. During the nonbreeding season (March to August), ovaries with no corpora lutea from six adult monkeys were used. Antral follicles 1000 µm in diameter were dissected from excised ovaries and processed as described previously [18]. Follicles showing obvious atresia (dark, dispersed granulosa cells) were discarded. Remaining follicles were held for 2–6 h in Tyrode-lactate (TL)-HEPES medium (37°C) until they were punctured for retrieval of germinal vesicle- (GV) stage oocytes.
Experiment 2. Oocytes from FSH-Stimulated Rhesus Monkeys Seven prepubertal rhesus monkeys (aged 23–25 mo, 2–3 kg), five adult rhesus monkeys (5–10 yr of age, 5–7 kg) in the breeding season, and 3 adult rhesus monkeys (4–15 yr of age) showing menstrual cycles during the nonbreeding season were used. Each animal received twice daily i.m. injections of 30 IU FSH (Neo-Fertinorm; Serono, France) for 8 days. For adult monkeys, the injections began on Days 1–3 of their menstrual cycles and for prepubertal monkeys on any day during April to July. Using a 20-gauge needle attached to a 5-ml syringe, 12–16 h following the last FSH injection oocytes were aspirated surgically from follicles ranging from 2.5 to 3.4 mm diameter into TL-HEPES medium [19] held at 37°C.
Oocyte In Vitro Maturation
For all monkeys, IVM was performed on oocytes that appeared normal (round and medium to lightly pigmented), contained an intact nuclear membrane (GV), and were enclosed by at least two layers of tightly condensed cumulus cells [20]. Essentially, oocytes were matured in modified CMRL-1066 [21] containing 20% heat-inactivated bovine calf serum (BCS; Hyclone Laboratories Inc., Logan, UT) 5 µg/ml ovine FSH (oFSH-NIADDK-NIH, AFP55518) and 10 ng/ml ovine LH (oLH-NIADDK-NIH, AFP4117A) for up to 36 h in 50-µl drops of medium under saline-equilibrated silicone oil at 37°C in a humidified atmosphere of 5% CO2 in air.
In Vitro Fertilization (IVF) and Embryo Culture
Oocytes that reached metaphase II (MII ova) were subjected to IVF. Either fresh semen collected by penile electroejaculation [22] or frozen-thawed spermatozoa, which have equal capacity for IVF as fresh spermatozoa [23], were used for insemination. Semen from one male was used throughout the study to minimize variability. Sperm capacitation and IVF were conducted as described previously [24]. Briefly, 20 x 106 washed motile spermatozoa/ml were resuspended in 2 ml TALP medium [17] overlaid with 2 ml silicone oil that was previously equilibrated with 5% CO2 in air. The spermatozoa were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 4–10 h, and then incubated for an additional 1–1.5 h in the presence of 1.0 mM each of caffeine and dibutyryl cAMP to induce hyperactivation. Hyperactivated spermatozoa were then diluted into 100-µl drops (2 x 105/ml final sperm concentration) of TALP medium supplemented with 2% BCS and containing MII ova. Spermatozoa and oocytes were coincubated for 12–16 h at 37°C in a humidified atmosphere of 5% CO2 in air and then ova were examined for evidence of fertilization. Ova exhibiting two pronuclei were cultured for embryo development in 50-µl drops (1–3 pronucleate ova per drop of mCMRL-1066 containing 20% BCS) for up to 11 days at 37°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2, being replaced into fresh culture medium every other day. Embryos were examined daily using Nomarski optics (200–400x magnification) on a Nikon Diaphot TMD microscope. The timing of blastocyst formation and hatching was recorded.
Data Analyses
Values for the proportions of IVM oocytes reaching MII were analyzed as percentages of total oocytes, and values for activated ova and all embryonic stages were analyzed as percentages of inseminated MII ova. All percentages were transformed by arcsine transformation prior to analyses. Group differences were detected by one-way ANOVA followed by least significant difference (LSD) test. Values with P < 0.05 were considered statistically different.
RESULTS
Experiment 1. Meiotic and Developmental Competence of Oocytes from Unstimulated Rhesus Monkeys
Table 1 shows the meiotic and developmental capacities of oocytes obtained during the breeding season from unstimulated prepubertal and adult rhesus monkeys. No significant differences were found in oocyte meiotic maturation, fertilization, or cleavage (P > 0.05) among the three groups. However, oocytes from prepubertal females displayed inferior development to the morula stage (4%, P < 0.05) compared with those from adult monkeys (18%, breeding season; 22% nonbreeding season). There was no significant difference between the proportions of ova reaching the morula stage between the two adult groups. Two blastocysts were obtained in one of five adult monkeys during the nonbreeding season.
|
Experiment 2. Effects of FSH Priming on Development of both Follicles and Oocytes of Prepubertal and Adult Rhesus Monkeys
A pool of follicles from prepubertal and adult rhesus monkeys in both the breeding and nonbreeding seasons responded to exogenous FSH treatment with rapid growth. However, the mean number of 2 mm follicles per female was lower in primed prepubertal vs. primed adult females in the breeding and nonbreeding seasons (16 ± 3 vs. 30 ± 5, respectively).
As shown in Table 2, oocytes from the three groups of females matured and underwent fertilization and the first cleavage in similar proportions. However, development beyond the eight-cell stage was significantly different among the three groups. Overall, oocytes from FSH-stimulated prepubertal females showed the greatest developmental potential followed by those from adult females primed during the breeding season. Oocytes from adult monkeys primed during the nonbreeding season displayed the poorest embryo development, only 3% morulae and 0% blastocysts. The highest proportions of inseminated MII oocytes reaching expanded and "hatched" (zona-escaped) blastocyst stages were also obtained in the FSH-primed prepubertal female group compared with the adult female groups that were primed during the breeding and nonbreeding seasons (expanded blastocyst, 19/43 [44%], 6/50 [12%], and 0/30 [0%]; hatched blastocyst, 14/43 [33%], 5/50 [10%], and 0/30 [0%], respectively; data not shown). Between the prepubertal and adult female groups, the time intervals postinsemination for blastocyst formation (166–180 h) and hatching (204–220 h) were similar. Blastocysts derived from the primed prepubertal females had a visible inner cell mass and could not be distinguished by size from those derived from adult monkeys. Figure 1 illustrates representative expanded and hatched blastocysts obtained from FSH-primed prepubertal rhesus monkeys.
|
|
DISCUSSION
Primate oocytes acquire meiotic and developmental potential relatively late in their growth process compared to other species [8]. Unlike in domestic animals and rodents, few blastocysts can be obtained from unstimulated primate oocytes matured and fertilized in vitro [14, 25]. To date, it is still unclear what signals initiate the events associated with competence to undergo embryo development to the blastocyst stage or when these events operate during oocyte maturation, although some preliminary results showed that in rhesus monkeys, acquisition of oocyte competence for embryogenesis is hormone-dependent [8, 14]. For the first time, our present study demonstrates that in unstimulated rhesus monkeys, oocyte competence increases with donor age. The effect of FSH on oocyte competence can vary depending on the animal's age and the breeding season.
In the present study, the effect of age on oocyte developmental competence was examined by comparing oocytes from unstimulated prepubertal (1–2 yr of age) females and from adults. The results showed that oocytes from prepubertal rhesus monkeys are less competent, as evidenced by less frequent morula formation than with oocytes from adult females in the breeding or nonbreeding seasons. This finding indicates that oocyte developmental competence is affected by age in primates, as in domestic animals [5, 11, 26] and rodents [1, 15]. In cattle and mice, oocytes acquire their competence in a gradual, age-dependent manner. Because we did not test the competence of oocytes from prepubertal females of different ages, it is unknown whether rhesus oocytes gradually acquire their competence with maturity of the donors. Nevertheless, our data suggest that in rhesus monkeys, puberty may be important for the occurrence of oocyte maturation events associated with embryo development competence.
Seasonal effects on several aspects of reproductive function in rhesus monkey are well known. Rhesus monkeys have reduced ovulatory and mating frequencies and fewer fertile copulations between May and September, whether the animals are feral, free-ranging, seminaturally housed, or maintained in controlled environments [27–30]. This period of reduced fertility is defined as the rhesus monkey's "nonbreeding season", despite that some females may continue to exhibit ovulatory menstrual cycles. Semen collected from males during this same period is usually of poor quality and unsuitable for IVF. Using cryopreserved rhesus monkey spermatozoa collected during the breeding season [23], we are able to examine IVM of rhesus monkey oocytes by IVF and examination of subsequent embryo development during the nonbreeding season. The results demonstrate similar meiotic and developmental competencies of oocytes collected from outdoor-housed females during the breeding and nonbreeding seasons, suggesting that there is no effect of season on oocyte competence in nonprimed females. This finding is very interesting because follicular development and hormonal profiles of adult rhesus females are different between the breeding season and nonbreeding seasons [29, 30].
As in immature mice and cattle [10, 11], in the present study, gonadotropin treatment of prepubertal rhesus monkeys increased not only the number of large antral follicles 2 mm, but also oocyte developmental competence. This result shows that prepubertal rhesus monkeys respond well to exogenous gonadotropins, consistent with the report by Wildt et al. in 1980 [31], who showed that GnRH infusion into infantile female rhesus monkeys could induce normal ovulatory menstrual cycles. However, in the present study, fewer follicles 2 mm were found in primed prepubertal females compared with FSH-stimulated adult monkeys (mean 16 ± 3 vs. 30 ± 5 per female, respectively). This may reflect that the immature macaque ovary has fewer follicles capable of responding to exogenous gonadotropins.
During the breeding season, the beneficial effect of FSH stimulation on oocyte developmental competence was apparent in both prepubertal and adult rhesus monkeys. In adult females primed with FSH, the frequency of blastocyst formation was 16%, which is higher than in a previous report [8]. However, oocytes from prepubertal monkeys exhibited an enhanced developmental response to FSH stimulation as judged by blastocyst formation (54%). The results suggested that the effect of FSH on oocyte competence was affected by animal age, which is consistent with earlier reports on mice [10] and cattle [11]. Oocytes from primed prepubertal monkeys showed greater developmental potential than those from primed adult monkeys during the breeding season, as evidenced by the higher percentage of blastocyst (54% vs. 16%), expanded blastocyst (44% vs. 12%), and hatched blastocyst (33% vs. 10%). This finding is particularly intriguing because it is inconsistent with previous studies on mice and cattle in which the competence of oocytes from FSH-primed immature animals is inferior to those from unstimulated or stimulated adult females [10, 11]. We propose that the higher developmental potential of oocytes from stimulated prepubertal monkeys might be related to the lack of dominant follicles. Several lines of evidence have shown that the dominant follicle produces an as-yet-unidentified direct dominance signal to influence other follicles, both on the same and on the contralateral ovary [32–34]. The suppressive effects of the dominant follicle involve not only the growth pattern of subordinate follicles, but also the competence of oocytes from both the subordinate follicles and the dominant follicle. Loss of dominance is an important factor in supporting or completing oocyte developmental competence [32]. The selection of the dominant follicle was recently believed to occur before the apparent decline in FSH levels or the appearance of any visible differences in follicle size [35, 36]. Thus, we propose that in adult monkeys, dominant follicles were selected and the dominance effect resulted in fewer oocytes capable of progressing to blastocyst, whereas in FSH-primed prepubertal monkeys, the greater proportion of blastocyst formation may reflect deficiency of the dominance mechanism.
Priming with FSH stimulated rapid growth of follicles in adult rhesus monkeys, both in the breeding and nonbreeding seasons. However, oocytes from adult females primed with FSH during the nonbreeding season showed significantly lower developmental competence than oocytes from primed adults during the breeding season. These findings suggest that 1) the processes of gonadotropin-stimulated follicular growth and oocyte development are separable in rhesus monkeys as in mice [10] and cattle [26], and 2) the action of exogenous FSH stimulation on oocyte competence is affected by season. Similarly, the ability of FSH to stimulate normal levels of estradiol production was also affected by the transition from the breeding to the nonbreeding season in rhesus monkeys [29]. Our study used three adult females exhibiting menstrual cycles during the nonbreeding season (March–August in Kunming, China). Previous studies have demonstrated differences of rhesus monkey menstrual cycles during the breeding and nonbreeding seasons. For example, menstrual cycles during summer showed depressed FSH levels in the follicular phase, markedly reduced progesterone levels in the luteal phase, and typically, altered follicular development [30]. Thus, we propose that the different endocrine profiles and follicular development patterns of menstrual cycles during the nonbreeding season are responsible for the altered responses of oocytes to follicular FSH stimulation. Of course, it is also possible that the superstimulation protocol utilized in the breeding season is not suitable for use in the nonbreeding season.
In summary, we conclude the following from the present study:
1. Rhesus monkey oocytes acquire their developmental competence in a manner that is dependent on the age of the donor female but not dependent on the season.
2. However, age and season both significantly affect the response of oocyte developmental competence to FSH stimulation in rhesus monkeys.
FOOTNOTES
First decision: 10 October 2000.
1 Supported by the Talent fund of Yunnan Province (96C0003G), China; projects under the Major State Basic Research Development Program, G2000016108; and is a Key Project of the Chinese Academy of Sciences KSCX1-05-02. Support was also provided by the National Institutes of Health (grants RR13439, HD-22023, and RO3-TW00840). 
2 Correspondence: Weizhi Ji, Director, The Kunming Institute of Zoology, The Chinese Academy of Science, 32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, People's Republic of China. FAX: 86 871 5139413; wji{at}mail.kiz.ac.cn
3 Current address: Barry D. Bavister, University of New Orleans, Department of Biological Sciences, and Audubon Institute Center for Research of Endangered Species, 14001 River Road, New Orleans, LA 70131.
Accepted: December 14, 2000.
Received: August 28, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  N. R Mtango and K. E Latham Differential Expression of Cell Cycle Genes in Rhesus Monkey Oocytes and Embryos of Different Developmental Potentials Biol Reprod, February 1, 2008; 78(2): 254 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zheng, W. Si, B. D. Bavister, J. Yang, C. Ding, and W. Ji 17{beta}-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys Hum. Reprod., October 1, 2003; 18(10): 2137 - 2144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Ptak, M. Tischner, N. Bernabo, and P. Loi Donor-Dependent Developmental Competence of Oocytes from Lambs Subjected to Repeated Hormonal Stimulation Biol Reprod, July 1, 2003; 69(1): 278 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. Davenport, C. J. Lees, H. L. Green, and K. A. Grant Long-Acting Depot Formulation of Luprolide Acetate as a Method of Hypothalamic Down Regulation for Controlled Ovarian Hyperstimulation and Oocyte Production in Macaca fascicularis Biol Reprod, June 1, 2003; 68(6): 2261 - 2266. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
