Maturation, Fertilization, and Development of Marmoset Monkey ...

BIOLOGY OF REPRODUCTION 56, 238-246 (1997)

Maturation, Fertilization, and Development of Marmoset Monkey Oocytes In Vitro Robert B. Gilchrist, Penelope L. Nayudu, and J. Keith Hodges Department of Reproductive Biology, German Primate Centre, G6ttingen, 37077 Germany ABSTRACT This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 tIm), class 2 (> 840-1000 m), class 3 (> 1000-1400 m), and class 4 (> 1400 tlm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II(MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development invitro. Oocyte meiotic competence and the time course of nuclear maturation are profoundly influenced by their follicular origin, and marginally by FSH treatment. INTRODUCTION When immature (germinal vesicle, GV) mammalian oocytes are artificially released from the follicular environment and cultured in vitro, they are able spontaneously to meiotically mature to metaphase II (MII, [1]). As the in vitro maturation (IVM) of oocytes has been shown to be a viable physiological phenomenon closely mimicking the in vivo process [2], this technique represents an alternative to superovulation as a means of obtaining functional ova. Combined with in vitro fertilization (IVF) and cryopreservation, oocyte IVM has important potential as a reproductive technology in assisted human fertility programs, in commercial production of embryos from domestic species, and in the conservation of endangered species and also as a means of obtaining ova/embryos for basic science studies

[3-5]. Successful implementation of IVM/IVF technologies, however, requires a thorough understanding of the specific physiological factors and in vitro culture conditions that are associated with high developmental potential of IVM oocytes. To date this information is available only in a few species of rodents and domestic ungulates and is essentially lacking in primates. What is understood of primate oocyte biology stems primarily from studies with Old World macaque species, while IVM of New World primate oocytes has been reported in only two species (marmoset [6, 7] and squirrel monkeys [8]). We have previously demonstrated the potential value of the marmoset monkey (Callithrix jacchus) as a model species for studies of primate follicular dynamics and oocyte biology [7, 9]. Nonovulatory antral follicles are particularly abundant in marmoset ovaries (-90 per ovary pair) and contain oocytes with an exceptionally high meiotic potential in vitro [7]. Marmoset oocyte germinal vesicle breakdown (GVB)- and MII-competencies are acquired appreciably earlier in follicular development than in oocytes from other primates [7, 10], and the high meiotic competence of oocytes from the smallest antral follicles may be related to the high degree of in vivo chromatin condensation and nucleolar compaction in these GV oocytes [9]. However, the developmental potential of IVM marmoset oocytes has not been previously examined, and there is to date only one report in any New World primate species. Yeoman et al. [8] demonstrated that oocyte meiotic and developmental capacity can be restored in aseasonal squirrel monkeys with in vivo FSH priming. Administration of FSH preparations has been shown to increase oocyte meiotic competence (rhesus [11], human [12]), and to modulate oocyte cytoplasmic maturation leading to notable increases in the developmental capacity of IVM oocytes (mouse [13], doe [14, 15], cow [16-18], ewe [19], and rhesus [11]). The primary objective of the present study was to establish whether marmoset oocytes matured in vitro were also cytoplasmically mature as evaluated by developmental competence. In order to achieve this, it was necessary to first establish techniques to capacitate sperm in vitro and develop an IVF procedure for IVM marmoset oocytes. Furthermore, examination of the time course of oocyte nuclear maturation in vitro was required, as this has not been previously accomplished in the marmoset or in any other New World primate species. A further aim of this study was to investigate the effects of in vivo FSH priming on the oocyte IVM competence and time course with reference to follicle size. MATERIALS AND METHODS Animal Stimulation and Oocyte Collection Ovarian cycles of adult female marmosets (1.8-5.1 yr of age) were monitored via plasma progesterone concentrations, and follicular phases were induced by controlled luteolysis (Day 0 = day of prostaglandin F2 ., Estrumate; Pitman-Moore, Burgwedel, Germany; [20]). From Day 0 to Day 3 of the follicular phase, animals received twice-daily

Accepted September 4, 1996. Received May 2, 1996. 'Correspondence and current address: Robert B.Gilchrist, Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, The University of Adelaide, Woodville, 5011, Australia. FAX: 61 8 82227521; e-mail: [email protected]

238

MEIOTIC AND DEVELOPMENTAL POTENTIAL OF MARMOSET OOCYTES i.m. injections of either 1) human (h) FSH (1.5 IU, Fertinorm HP75; Serono, Unterschleissheim, Germany; contains < 0.001 IU LH; n = 5) or 2) control (normal saline; n = 5). The treatment represents 1.2 IU hFSH/day per kg, which is comparable to the typical dosage used in rhesus monkey stimulation [11, 21] and twice that generally used in human IVF stimulation protocols. Ovary pairs were collected on the morning of Day 4 of the follicular phase, and antral follicles were excised and separated into classes according to size in Leibovitz L-15 medium (Gibco, Berlin, Germany) with 5% heat-inactivated fetal bovine serum (Sigma, Deisendorf, Germany). Follicles classified as small antral in a previous study [7] were further divided into class 1 (660840 jim) and class 2 (> 840-1000 Rim) follicles, and large antral follicles into class 3 (> 1000-1400 jim) and class 4 (> 1400 jim). Degenerated and highly atretic follicles (dark, uneven granulosa and theca cells), found only in class 1 follicles, were discarded. Follicles were punctured and oocytes were collected and classified as 1) cumulusenclosed oocyte (CEO; a minimum of one complete layer of closely associated cumulus cells) or 2) partially naked and naked oocytes (less than one layer of cumulus cells or loosely attached cumulus cells) as described previously [7]. Oocyte IVM Oocyte culture conditions were modified from those previously used for marmoset [7] and rhesus oocytes [11, 22]. Eight hundred and twenty-three naked oocytes and CEOs were cultured in 100-p1l droplets of Waymouth Medium MB752/1 (Gibco) supplemented with 1 jg/ml (5.8 IU/ml) hFSH, 10 jig/ml (115.6 IU/mI) hLH, 1 ig/ml estradiol (Sigma), 20% fetal bovine serum (Sigma), 0.5 mM sodium pyruvate (Sigma), 1 mM glutamine (Gibco), 10 mM sodium lactate (Sigma), 4 mM hypotaurine (Sigma), 66 mg/L penicillin G-K salts (Sigma), and 50 mg/L gentamycin sulfate (Sigma) under nonwashed silicon oil (Aldrich, Steinheim, Germany) in 25-mm petri culture dishes (Falcon, Plymouth, England) in a humidified atmosphere of 5% CO2 in air. To determine timing of nuclear maturation, cumulus cells were mechanically/enzymatically removed using a fine-bore micropipette in maturation medium supplemented with 500 ,ug/ml hyaluronidase (Sigma) and 10 ig/ml soybean trypsin inhibitor (ICN, Meckenheim, Germany) under oil after 22 ± 1 h IVM. Oocytes were then transferred to a clean droplet and carefully observed for the presence of a polar body (PB) by rolling the oocyte within the droplet under an inverted Zeiss Axiovert 405M microscope (Carl Zeiss, Gottingen, Germany) equipped with a chamber gassed with 5% CO2 in air (37°C). After 24, 32, and 40 1 h in culture, PB-stage oocytes were transferred to IVF droplets. Oocytes remaining at 48 1 h were fixed, and after the chromatin was stained (Hoechst 33258) and the microtubulin was labelled (anti-ea-tubulin/fluorescein isothiocyanate), they were mounted as previously reported [7] to assess meiotic progression using fluorescence microscopy. Sperm Collection and Preparation Epididymal and ejaculated sperm were collected from males of proven fertility. Epididymides were obtained from males that had been separated from their female partners for 4 days before castration, and were collected in modified Tyrode's albumin lactate pyruvate (TALP)-Hepes medium (TL medium supplemented with 3 mg/ml BSA [Sigma, fraction V], 0.25 mM sodium pyruvate, and 10 mM Hepes [Sigma]; [23]) at 30°C. Epididymides were separated from

239

the testis, and the vas deferens and cauda epididymides were dissected free of fat, connective tissue, and capillaries. After washing in TALP capacitation/IVF medium (TALP medium with 0.5 mM glutamine and 50 jig/ml gentamycin) and transfer to droplets of 200 R1 of preincubated medium under silicon oil, sperm were gently squeezed from the vas deferens and cauda epididymidis. The resulting sperm suspension was incubated for 15-20 min (37°C, 5% CO 2 in air). Ejaculated sperm from 2-3 male marmosets were collected by vaginal washing [24]. Briefly, males were separately introduced into the cages of ovariectomized females and when copulation was observed, the female was removed, and the vagina was gently, repeatedly rinsed with 300 I of TALP-Hepes, allowing collection of the ejaculate. The two sperm suspensions (epididymal and ejaculated) were then prepared for IVF identically and in parallel using a protocol modified from rhesus IVF [23]. Preequilibrated TALP medium (200 ,ul) in an Eppendorf centrifuge tube 1 was underlaid with -200 Il sperm suspension. After 300 to 45-min incubation at 37 C to allow the sperm to swim up, the top 300 was removed. Sperm (4 x 106) were resuspended in 200 il of preequilibrated TALP medium under oil; and after 2-h preincubation, 100 M each of caffeine (Sigma) and dibutyryl cAMP (dbcAMP; Sigma) were added to induce hyperactivation. In each experiment, leftover sperm suspension was utilized to examine the effects of caffeine/dbcAMP on various sperm characteristics in vitro. After 2-h preincubation, capacitation droplets were supplemented with either 0 (control), 10, 100, or 1000 jIM of each of caffeine and dbcAMP, and after various incubation intervals the sperm were diluted (1:100) and scored under the inverted microscope for motility, longevity, hyperactivity, and vigor. The choice of sperm conditions used for IVF was based on these observations of ejaculated and epididymal sperm in vitro and on preliminary IVF investigations. IVF and Embryo Culture After 2- to 3-h incubation with caffeine/dbcAMP, 10 lI was removed from the mid-outer portion of the capacitation droplet and added to 40-p1 droplets of preincubated IVF medium (TALP; 4 x 106 sperm/ml final) containing IVM oocytes. A total of 148 cumulus cell-denuded IVM (MII) oocytes from 3 animals (2 FSH-primed, 1 nonstimulated) were coincubated with sperm for 18-24 h (37°C, 5% CO 2 in air). Thereafter, oocytes/embryos were freed of loosely bound sperm by gentle micropipetting, transferred to 100pl droplets of embryo culture medium under silicon oil, and observed under an inverted microscope for the presence of pronuclei. Embryos were cultured for up to 2 wk in a humidified atmosphere of 5% C0 2:10% 02:85% N2 (37°C) using a modified two-step embryo culture medium [11, 22]: 1) TL medium with 3 mg/ml BSA, 10% fetal calf serum, 0.5 mM sodium pyruvate, 1 mM hypotaurine, 1 mM glutamine, and 50 pxg/ml gentamycin for the first 48 h; and thereafter 2) CMRL-1066 (Gibco) medium with 20% fetal calf serum, 0.5 mM sodium pyruvate, 10 mM sodium lactate, 1 mM glutamine, I mM hypotaurine, 66 jig/ml penicillin G-K salts, and 50 jig/ml gentamycin. Embryos were examined daily, and those exhibiting no developmental change for the previous 48 h were fixed for blastomere counts using fluorescence microscopy as described above. The embryo developmental stage reached was determined retrospectively based on the number of blastomeres. Par-

240

GILCHRIST ET AL. r

thenogenetic control oocytes (20 MII oocytes from 8 animals) were handled as described above except that sperm were not added to the insemination droplet.

Control animals (n=5; 455 oocytes) I

FSH-primed animals (n=5; 440 oocytes)

100

100

80

Data Analyses

80

0 60

60

40

40

20

20 0

0

1

2

3

4

Follicle Class FIG. 1. Oocyte-cumulus cell associations in relation to FSH priming and follicle size. Columns represent percentages of total oocytes enclosed in cumulus cells at collection from a given follicle class: 1 (660-840 jm), 2 (> 840-1000 jim), 3 (> 1000-1400 jim), and 4 (> 1400 jIm). Oocytecumulus cell status was highly dependent on follicular origin but unaffected by FSH priming of animals. Class 4 oocytes from nonstimulated animals were excluded from statistical analysis because of low numbers (n = 3). Different letters denote significant differences between treatments and follicle classes (p < 0.05).

Analysis of variance using generalized linear models analysis (randomized block design; [25]) was employed to examine the relationships between the following factors: 1) oocyte-cumulus cell associations between follicle size groups and treatment (Fig. 1), 2) oocyte meiotic competencies (Fig. 2) between different follicle sizes/animal treatments, and 3) rate of meiotic maturation in relation to time cultured in vitro, follicle size, and animal treatment (Figs. 3 and 4). To allow for unequal variances, an arc sine transformation of percentages (weight; total number of oocytes per animal) was conducted and a Freeman Tukey transformation for low numbers of observations was carried out, and treatment differences were then detected by comparing least squares means. A p value < 0.05 was considered statistically significant. RESULTS In Vivo Oocyte-Cumulus Cell Status The proportion of oocytes enclosed in cumulus cells increased progressively with follicular size (p < 0.0001) and was unaffected by FSH priming (Fig. 1). Significantly (p < 0.01) fewer oocytes from class 1 follicles were cumulus enclosed (50.2% and 52.3% from control and treated animals, respectively) as compared to oocytes from all other classes. More than 90% of oocytes from follicles > 1 mm (classes 3 and 4) were classified as CEOs. Oocyte Meiotic Competence

Cumulus Enclosed-

+ -

Follicle Class

+ ++

FSH Priming - _ I -

-

+ -

- _

+

-

+ -

+ +

-

_

22-

-

+

+ + 3 --

-+ -

-

+ +

4 -

FIG. 2. Meiotic resumption and maturation of oocytes in relation to follicle size, FSH priming, and oocyte-cumulus cell associations. Columns represent percentages of oocytes undergoing GVB (A) and reaching MII (B)after 48 h in vitro. CEOs and naked oocytes were collected from FSHprimed and control animals (n = 10) and separated according to antral follicle size: class 1 (640-840 jim), class 2 (> 840-1000 jim), class 3 (> 1000-1400 i.m), and class 4 (> 1400 jim). Oocyte GVB- and MII-competencies increased notably with follicle size and slightly with FSH priming and were unaffected by the presence of cumulus cells. Columns with differing letters within a graph are significantly different (p < 0.05). Total numbers of oocytes (n = 10 animals) making up column (left to right) percentages: 106, 113, 72, 92, 30, 101, 28, 94, 52, 8, 76, 15. Missing columns represent a lack of follicles/oocytes in class, and data from the column without a letter were excluded from statistical analysis owing to low numbers.

Oocyte GVB- and MII-competencies increased notably with follicular size (p < 0.01 and p < 0.0001, respectively) and were independent of oocyte-cumulus cell associations. FSH priming of animals slightly increased competence of oocytes to resume and complete meiosis in vitro (p < 0.01 and p < 0.05, respectively). A total of 790 oocytes were cultured in vitro; among these, 96% (524 of 546) of the CEOs resumed and 77% (421 of 546) completed meiotic maturation. Regardless of FSH treatment or cumulus cell status, oocytes from class 1 follicles exhibited significantly lower GVB-(p < 0.05) and MII-competencies (p < 0.0001) than oocytes from all other classes (Fig. 2). Maximum GVB- and MII-frequencies (CEOs; 95-100% and 82100%, respectively) were attained in oocytes from class 2 follicles (> 0.84-1 mm), with no further increases in oocytes from follicles > I mm (classes 3 and 4; Fig. 2). The most notable effect of the FSH priming was on oocytes from class 1 follicles, in which GVB competence (but not MII competence) was significantly (p < 0.05) increased in both naked oocytes and CEOs. The presence of cumulus cells tended to be associated with higher GVB- and MIIcompetencies, but the differences were not significant. Sixty oocytes (7.6% of total oocytes) remained meiotically arrested (29 of which originated from the naked class I category from control animals); among these, 14% presented dispersed GV chromatin configurations, 31% intermediate chromatin, and 55% highly condensed chromatin. Twentytwo oocytes (2.7%) degenerated in vitro, and these came almost entirely (95%) from class 1 follicles.

MEIOTIC AND DEVELOPMENTAL POTENTIAL OF MARMOSET OOCYTES

241

Rate of Oocyte Nuclear Maturation Of the MII-competent oocytes from control animals, 68.9% of CEOs and 63.2% of naked oocytes completed meiotic maturation within 24 h in vitro, and FSH priming of animals increased these frequencies in CEOs (p < 0.05, 84.0%) and in naked oocytes (not significant, 71.0%; Fig. 3). The proportion of mature oocytes continued to increase (p < 0.001) between 24 and 32 h in vitro (range, 82-96%), with a further marginal increase after 33 h such that most (91-99%) of the MII-competent oocytes were mature by 40 h in vitro. In general, the rate (speed) of meiotic maturation was significantly higher with FSH priming (p < 0.01), with increasing follicle size (p < 0.01), and with the association of cumulus cells (p < 0.01). In relation to each factor, the differences in rate of meiotic maturation were most evident at 24 h; they diminished with increasing time in vitro as oocytes that matured more slowly reached MII and the total proportion mature neared 100%. In untreated animals the rate of meiotic maturation of CEOs was progressively faster (p < 0.05) with increasing follicle size (Fig. 4). However, the effect of follicle size on rate of nuclear maturation was eliminated when animals were primed with FSH. That is, from primed animals, class 1 CEOs matured as fast as those from classes 2 and 3. The more advanced rate of oocyte meiosis from primed compared with control animals was consistent in all three follicle-size classes. Therefore, the effect of increased in vivo FSH was most pronounced (p < 0.01) in the smallest antral follicles (class 1). In general, by 32 h in vitro, differences in the rate of maturation of oocytes from different follicle classes persisted; however, the differences were lessened and were not significant (data not shown). The single exception to the trend was in CEOs from the largest follicles (class 4) from primed animals. Oocytes from this largest size class presented a lower rate of maturation, with 67%, 80%, and 100% being mature after 24, 32, and 40 h, respectively. Sperm Characteristics In Vitro Both ejaculated and epididymal sperm responded to dbcAMP/caffeine in vitro by displaying hyperactive motility characterized by vigorous, nonlinear motion, associated with high-amplitude lateral head movement. Ejaculated sperm exhibited high-velocity, discontinuous motility in vitro, and notable hyperactivity and rapid loss of motility in response to dbcAMP/caffeine. Epididymal sperm were less sensitive than ejaculated sperm to stimulants, displaying greater longevity than ejaculated sperm under the same conditions. Epididymal sperm showed progressive increases in hyperactivity with increasing concentrations of dbcAMP/caffeine. While epididymal sperm exposed to 10 RIM caffeine/dbcAMP had motility patterns similar to those of control sperm, moderate hyperactivity was observed 26 h after exposure to 100 M. Within 1 h of exposure to 1 mM caffeine/dbcAMP, sperm exhibited extreme hyperactivity, and within 4-6 h they generally showed complete loss of motility. This range of in vitro capacitating conditions proved unsuitable for ejaculated sperm even though motile hyperactivated sperm could be obtained, as fertilization and development frequencies of IVM oocytes were higher with use of epididymal compared to ejaculated sperm (Table 1). On the basis of these preliminary experiments, further IVF experiments were conducted using epididymal sperm capacitated with 100 IM dbcAMP and caffeine.

FIG. 3. Rate (speed) of meiotic maturation in vitro in relation to FSH priming and oocyte-cumulus cell associations. Columns represent percentages of MII-competent oocytes (*) having reached Ml after 24, 32, and 40 h in vitro. Rate of meiotic maturation was higher with FSH priming and was marginally (nonsignificantly) increased with the association of cumulus cells, these effects being most evident after 24 h. Columns with differing letters are significantly different (p < 0.05). Data include naked ooytes and CEOs from antral follicles > 660 p.m from 5 control (n = 76 and 196 oocytes, respectively) and 5 FSH-primed animals (n = 69 and 225 oocytes, respectively).

IVF and Embryo Development Mature oocytes from three animals were incubated with sperm to establish whether IVM oocytes could be successfully fertilized and whether they possessed embryonic developmental potential. A high proportion (average 84%) of IVM oocytes from nonstimulated and FSH-primed marmosets were fertilized in vitro using epididymal sperm. Of these, 27% developed to morula- and 4% to blastocyststage embryos (Table 2 and Fig. 5). Oocytes from all follicle/cumulus cell classes exhibited fertilization and developmental competence. Embryos derived from oocytes from the smallest antral follicles (class 1) readily developed to the morula stage (25% and 28% from both naked and CEOs, respectively), including 1 naked oocyte from a nonstimulated animal that developed to the hatched blastocyst 100

100 b,c

ib,c

iC

80 e4 eq

60

aPb .. ..........

40

_-A.. -| FSH-Primed

-

a 20

b~

80 . b,c ........... ........ ; 60 ............

.................

..............

0 1

40

Control 20 0 2

3

4

Follicle Class FIG. 4. Effects of follicle size and FSH priming on rate (speed) of meiotic maturation after 24 h in vitro. Points represent percentages of MII-competent oocytes (CEOs only) mature after 24-h IVM from either control or FSH-primed animals from different follicle size classes: 1 (660-840 .m), 2 (> 840-1000 Ipm), 3 (> 1000-1400 Ipm), and 4 (> 1400 Itm). In control animals, oocytes from larger follicles required less time in vitro to achieve MII than oocytes from smaller follicles; however, FSH priming of animals overcame this effect of follicle size. After 24 h in vitro, CEOs from FSH-primed animals were more meiotically advanced than their control counterparts from the same-sized follicles. Different letters denote significant differences between treatments and follicle classes (p < 0.05).

242

GILCHRIST ET AL. TABLE 1. Comparison of fertilization and development frequencies of oocytes inseminated with epididymal or ejaculated sperm." Sperm source

nl'

Fert.

- 2C

Epididymal Ejaculated

44 33

41 (93) 11 (33)

39 (89) 10 (30)

Number at developmental stage (%/, - 5-8 C - 9-16 C - 3-4 C 37 (84) 10 (30)

33 (75) 8 (24)

26 (59) 4 (12)

Morula

Blasto.

23 (52) 3 (9)

3 (7) 0 (0)

' CEOs from 2 FSH-primed animals were matured in vitro and randomly allocated to in vitro insemination with either epididymal or ejaculated sperm previously incubated for 2 h in the presence of 100 pIM each of dbcAMP and caffeine. C, cell; Fert., fertilized; Blasto., blastocyst. '' Number of inseminated MII oocytes. Percentage of inseminated MII oocytes.

stage. None (0 of 20) of the parthenogenetic control oocytes activated. Naked oocytes and CEOs exhibited comparable fertilization and developmental potentials. Regardless of animal treatment or follicle/cumulus cell class, the vast majority of embryos exhibited pronounced blastomere swelling (obliterated perivitelline space, poorly discernible cleavage divisions) by the 4-cell stage with blastomeres resuming a rounded shape between the 8- and 16-cell stages (developmental progression could be monitored by counting blastomere nuclei). The following rates of embryonic development were observed: pronuclei, by 18 h postinsemination; 2 cell, Days 1-1.5; 4 cell, Days 2-3; 8 cell, Days 2-4; 16 cell, Days 4-5; compacting morula, Days 5-8; blastocyst, Days 7-10; expanded blastocyst, Days 11-13; and hatched blastocyst, Day 13. A high proportion of advanced morulae (> 80 blastomeres) arrested without showing signs of blastocele formation. DISCUSSION The results of this study have shown that the maturation potential of marmoset oocytes in vitro is closely related to follicle size, that it can be only marginally improved with the in vivo FSH-priming protocol used, and that IVM oocytes can achieve advanced stages of preimplantation embryonic development in vitro. In oocytes from untreated marmosets, the competence to resume and complete meiosis and the time course of nuclear progression in vitro was highly dependent on the size of the follicle from which the oocyte originated. Marmoset oocytes from intermediatesized antral follicles exhibit a notably higher capacity to meiotically mature in vitro (90-100%, [7] and present study) than oocytes from macaques (15-56%, [10, 26-28]) and humans (17-68%, [12, 29-32]). In unprimed marmo-

sets, oocytes from the smallest antral follicles have a lower meiotic potential than oocytes from larger antral follicles, and those that are able to mature do so more slowly. The importance of follicle size in determining oocyte maturation potential has been comprehensively documented in many species (reviewed in [33, 34]), including primates (present study; [6, 7, 10, 27, 28]). These studies illustrate that events occurring in the later stages of antral development modulate oocyte cytoplasmic maturation, expressed in a higher rate (speed) of meiosis, a higher capacity to complete meiosis, and enhanced developmental potential. Although the exact nature of ooplasmic events occurring in larger antral follicles remains poorly understood, the association of distinct morphological events, such as oocyte nucleolar compaction and the condensation of chromatin, may play a functional role in the acquisition of oocyte meiotic and developmental potentials [35-37]. Compared to oocytes from other primates [10, 38-40], marmoset oocytes from small antral follicles exhibit very high degrees of chromatin condensation and nucleolar compaction [9], and this may partly explain the high meiotic potential in this species. It is hypothesized that progressive in vivo chromatin condensation with follicular development correlates with, and is a prerequisite for, MII competence of oocytes [35, 37]. Furthermore, it is believed that cessation of oocyte rRNA synthesis, as evidenced by nucleolar compaction [41-43], may correspond to the acquisition of sufficient "translation machinery" necessary for the synthesis of proteins regulating meiosis [36]. The relationship between follicle size and oocyte maturation potential can also be examined in terms of the timing of nuclear progression. In nontreated marmosets, CEOs from larger antral follicles matured faster than their small

TABLE 2. Fertilization and development of IVM oocytes in relation to follicle size and oocyte-cumulus cell associations. Follicle class, 1 2 3 4 Total

C.C. status'

Number at developmental stage (%)' - 9-15 C - Morula - 5-8 C - 2-4 C

Blasto.

7 (39) 16(41) 5(31) 4

5 (28) 13 (33) 1 (6) 3

1 (3)' 1 (6) 1

16 (44) 49 (62)

10 (28) 32 (41)

9 (25) 22 (28)

2 (6)' 3 (4)

65 (57)

42 (37)

31 (27)

5(4)

n

-> Fert.

CEO CEO CEO CEO

18 39 16 6

14 (78) 36 (92) 14(88) 5

12 (67) 36(92) 13(81) 5

8 (44) 28 (72) 9(56) 4

Naked CEO

36 79

28 (78) 69 (87)

27 (75) 66 (84)

115

97(84)

93 (81)

' Class 1 (640-840 itm), class 2 (> 840-1000 pim), class 3 (> 1000 1400 tm) and class 4 (> 1400 im). ' C.C., cumulus cell; Naked, naked or partially naked oocyte at collection. 'Number of inseminated MII oocytes. Data were pooled from 2 FSH-primed and 1 control animal using epididymal sperm. d Percentage of inseminated MII oocytes. C, cell; Fert., fertilized; Blasto., blastocyst. One blastocyst hatched.

MEIOTIC AND DEVELOPMENTAL POTENTIAL OF MARMOSET OOCYTES

243

FIG. 5. Marmoset monkey oocytes after maturation, fertilization, and development in vitro. A) Representative IVM (MII) oocyte with PB after 24 h in vitro. B) A 1-cell embryo with 2 pronuclei and 2 PBs (1 PB is slightly out of focus) at 24 h postinsemination (p.i.). C) A 6-cell embryo at 1.5 days p.i., originating from a CEO from a large antral follicle (> 1.4-2 mm), that developed a blastocele at 9 days p.i. (D) and went on to an expanded blastocyst, collapsing on Day 12 p.i. with -140 blastomeres. E)A hatched blastocyst at 13 days p.i. with -450 blastomeres, originating from a small antral follicle (> 0.84-1.0 mm). F) Fluorescent micrograph (DNA fluorochrome Hoechst 33258) of a 4-cell embryo presenting the nuclei of 3 blastomeres and numerous fluorescing sperm heads bound to the zona pellucida at a different focal level. All micrographs were taken on a Zeiss Axiovert 405M microscope (objectives: A-D, x40; E, x20; F, X100).

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antral counterparts, as has also been observed in pig [36] and rabbit oocytes [44]. This would support the notion that oocytes from small antral follicles, despite having MII competence, are less cytoplasmically mature than those from larger follicles. Studies with bovine oocytes illustrate that oocytes maturing faster in vitro are more cytoplasmically mature than slow-maturing oocytes, as evidenced by enhanced developmental competence after IVF [45]. The time course of oocyte nuclear maturation in vitro has not been previously investigated in a New World primate species. The present study has shown that marmoset oocytes require appreciably less time in vitro to mature (20-24 h) than do oocytes from rhesus monkeys (30-34 h, [27, 46]) and humans (36-47 h, [32, 47]); this time period is more comparable to that required by oocytes of domestic ungulates (16-27 h, [45, 48-51]). Oocytes from the largest follicles (> 1.4 mm; found only in FSH-primed marmosets) presented an anomaly in the relationship between follicle size and the timing of meiotic maturation in that they tended to mature more slowly than those from smaller follicles, although this was nonsignificant because of the small numbers of oocytes in this class. This difference is important, as these oocytes come putatively from the most "normal" follicles, i.e., those that are most likely to ovulate. The increased time required to meiotically mature may be explained by slower withdrawal of the cumulus-cytoplasmic processes, which are known to mediate meiotic arrest [52], from these oocytes. Oocytes from the largest follicles have appreciably more cumulus layers than those from smaller follicles, and the cumulus layers further differ in that they undergo some degree of mucification and expansion during IVM, rather than the more rapid attachment and compaction typical of the cumulus mass of oocytes from smaller follicles. Furthermore, remnants of the corona radiata in the zona pellucida following cumulus cell removal after 24-h IVM are seen only in oocytes from the largest follicles. These observations indicate that these oocytes remain in metabolic contact with the cumulus cells for a longer period. In vivo FSH priming of marmosets had subtle effects on oocyte biology, slightly improving GVB- and MII-competencies and significantly decreasing the time required to meiotically mature, implying an FSH-induced improvement in nucleocytoplasmic interaction. Administration of FSH to mice [13], domestic ungulates [14-19], and primates [8, 11, 12] has previously demonstrated the essential role FSH plays in orchestrating follicular events that modulate oocyte cytoplasmic maturation-regulating oocyte meiotic and developmental capacity. In the present study, the effects of in vitro FSH exposure were not equivalent to in vivo administration, as oocytes from unprimed animals showed lower meiotic potential than those from primed animals in the presence of constant FSH/LH levels during IVM. The most notable effect of in vivo FSH priming was on the smallest antral follicles-increasing GVB competence and rate of meiotic maturation (but not MII potential) to the values seen in oocytes from the largest follicles. This implies that in nonstimulated animals the in vivo supply of FSH to small, but not large, antral follicles is a limiting factor. This also supports the notion that the larger antral follicles are more sensitive to and better able to sequester the circulating FSH in nonstimulated animals (reviewed in [53]). FSH priming is hence a logical experimental step to alleviate the FSH deficiency and thus improve the meiotic and developmental potential of oocytes from small antral follicles, which are particularly abundant in the marmoset.

Despite the subtle effects of FSH priming on oocyte function, pronounced follicle recruitment and growth (results not shown) or increases in the proportion of CEOs, often observed in primates [11, 21,54, 55] and other species [13, 17] in response to FSH administration, were not observed in this study. The reason for this limited ovarian response to FSH administration is unknown. Marmoset FSH receptors respond to hFSH, as demonstrated by cell proliferation and estrogen production by isolated granulosa cells in vitro [56]. The relatively low ovarian response observed in these experiments may be attributable to in vivo structural modifications to the gonadotropin molecule that reduce its in vivo bioactivity and/or clearance rate from the circulation. Alternatively, in the absence of information about the endogenous FSH profiles, the possibility exists that the timing of FSH administration was inappropriate. Increases in the proportion of CEOs and in the number of layers of cumulus cells surrounding oocytes, in association with increasing follicular size as found in this investigation and other studies [17, 35, 51], indicate that the quality and maturity of the oocyte-cumulus cell complex increases with follicle size. The smallest antral follicles in marmosets appear the most atretic and yield the highest proportion of partially naked oocytes (-50%), many of which resume meiosis in vivo [9] and present meiotic and spindle abnormalities after IVM [7]. Accordingly, partially naked oocytes from the smallest antral follicles were more meiotically advanced after 24 h in vitro than their cumulusenclosed counterparts (present study, results not shown). However, in contrast to the results obtained with rhesus oocytes [46], and similar to observations with ungulate oocytes [50, 51], partially naked oocytes matured after 48 h in vitro with the same frequencies as CEOs. Furthermore, naked oocytes exhibited fertilization and developmental potentials comparable to those of CEOs. This has an important practical implication in that, as also reported for bovine oocytes [57], oocytes having incomplete cumulus cell layers and showing moderate degrees of atresia at collection should not be discarded, since a high proportion can be fertilized in vitro and retain developmental potential. The possibility exists, however, that these embryos could contain chromosomal abnormalities, in light of our previous report of spindle abnormalities from this group [7]. The successful fertilization of IVM oocytes and subsequent embryo development demonstrate that a high proportion of marmoset oocytes meiotically matured in vitro are also cytoplasmically mature. Previous reports of in vitro fertilization and embryo development in the marmoset have utilized in vivo-matured oocytes aspirated from preovulatory follicles [58, 59], and attempts to fertilize oocytes from nonovulatory follicles were unsuccessful. In contrast, the present study has shown that a high proportion of oocytes from all sizes of nonovulatory antral follicles have developmental potential after IVM/IVF, including oocytes from small antral follicles (0.66-0.84 mm), at which stage MII competence is first acquired [7]. Further studies with larger numbers of oocytes are required to determine whether marmoset oocyte developmental potential increases with follicular size or with FSH priming, as has been demonstrated for rhesus oocytes [11, 28] and those of other species [1219, 34]. This study also describes a procedure for IVF of IVM marmoset oocytes that was adapted from the method used for rhesus oocytes [11, 22]. Preliminary attempts to utilize naturally ejaculated sperm for IVF were unsuccessful. Although ejaculated sperm could be reliably collected using

MEIOTIC AND DEVELOPMENTAL POTENTIAL OF MARMOSET OOCYTES vaginal washing [24], these sperm were extremely sensitive to handling procedures and the culture conditions used, and fertilization rates were very low. In contrast, epididymal sperm responded in a dose-dependent manner to caffeine and dbcAMP by displaying hyperactivation, and they exhibited higher fertilization frequencies than ejaculated sperm. Under the conditions used in this experiment, the most suitable epididymal sperm for IVF were obtained with 100 M caffeine/dbcAMP, which is intermediate between the concentrations previously used for marmoset (10 pIM, [58, 59]) and rhesus sperm (1 mM, [22]). However, further work is required to fully optimize sperm capacitation conditions. In contrast to previous studies reporting IVF of New World primate oocytes [8, 59], in the present investigation relatively high proportions of embryos obtained from IVM/ IVF marmoset oocytes reached advanced preimplantation development stages in vitro. While a high proportion of embryos reached the advanced morula stage (> 80 blastomeres), many of these embryos failed to progress to blastocele formation, as also observed for in vitro-produced (IVP) rhesus embryos [11]. The pronounced blastomere swelling bserved throughout the early cleavage stages in this study may be an indication of the suboptimal quality of IVM oocytes, and/or it may be an artefact caused by inappropriate culture conditions. The developmental potential of embryos that exhibited blastomere swelling was not necessarily compromised, however, as some of these embryos went on to develop to the blastocyst stage. Furthermore, these embryos achieved advanced developmental stages only slightly more slowly than reported for in vivofertilized and -developed embryos (hatched blastocyst: IVM, Day 13; in vivo matured, Days 10-11 [60, 61]). Blastomeres of IVP rhesus embryos also swell with use of the same embryo culture medium (R.D. Schramm, personal communication), although less severely than observed in this study. This is the first report of IVM/IVF oocytes from an untreated New World primate, and the second in any primate species to demonstrate embryonic development to the hatched blastocyst stage in vitro from IVM oocytes. Although the in vitro production of rodent and ruminant embryos is now commonplace [5], fertilization and development of IVM oocytes from primates has been achieved in only five species (marmoset, present study; squirrel monkey [8]; rhesus monkey [11, 28, 62]; pig-tailed macaque [63]; human [30-32]). As superovulatory techniques are not available in Callitrichidae species, IVM of oocytes from excised ovaries is currently the only means available for obtaining moderate numbers of functional ova. This investigation illustrates that the numerous oocytes that can be obtained from excised marmoset ovaries (-90 per ovary pair) can be matured in vitro and used as a source of ova (-65 per ovary pair) for IVP marmoset embryos. However, attempts to improve the maturation potential of marmoset oocytes with in vivo administration of relatively high doses of hFSH met with minimal success. Oocyte meiotic potential was primarily determined by the follicular origin of the oocyte. Oocytes from the smallest antral follicles were distinct from those from larger follicles in terms of reduced cumulus cell associations, rate of nuclear maturation, and MII competence, although a proportion exhibited advanced developmental potential after IVE While the procedures described in this study may be suitable for IVM of marmoset oocytes, further investigations are required to improve in

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vitro embryo culture and particularly sperm capacitation conditions. ACKNOWLEDGMENTS The authors wish to thank Petra Kiesel for assistance with follicle dissection, Nicole Niisse and Susanne Rensing for surgery and bleeding of animals, Conny Casper and Dorothea Lindner for animal maintenance and husbandry, and Manzoor A. Nowshari for details of epididymides preparation. We are especially grateful to Drs. R. Dee Schramm and Barry D. Bavister for providing the opportunity to learn nonhuman primate oocyte IVM/IVF procedures in their laboratories. The pituitary gonadotropins used were generously provided by the National Hormone and Pituitary Program.

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