Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O₂) or high (20% O₂) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO₂, and 5% or 20% O₂. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O₂) and 11.78 ± 1.06 μm (20% O₂) on Day 4 and 20.94 ± 1.24 μm (5% O₂) and 19.12 ± 1.36 μm (20% O₂) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O₂ (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O₂) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (≥150 μm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O₂ concentrations (5% and 20% O₂). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O₂ concentrations (P < 0.05). When the O₂ tensions were compared to each other in the different days of culture, 20% O₂ was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O₂ (P < 0.05). However, follicles cultured with 5% O₂ had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O₂ (P < 0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O₂ tensions. However, only oocytes (16.7%) from follicles cultured in 20% O₂ resumed meiosis. In conclusion, concentration of 20% O₂ was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.     

In vitro growth of oocytes from primordial follicles isolated from frozen-thawed lamb ovaries

A study was conducted to develop an in vitro culture system for growing sheep oocytes from isolated primordial follicles. Enzymatically isolated neonatal sheep primordial follicles were cultured in Waymouth MB752/1 medium containing BSA (3 mg/ml) + ITS (1%, v/v) over 28 days. In Experiment 1, primordial follicles (average diameter 40.2+/-0.60 microm) were cultured at densities of 20, 50 and 100 follicles per well. Less than 20% of the oocytes survived to day 28 but there was a significant (P < 0.05) increase in median oocyte diameter from day 2 to day 28 for oocytes cultured at the higher densities of 50 and 100 follicles. In Experiment 2, two methods to improve oocyte:granulosa cell associations were tested. Altering the fibronectin coating regime did not improve oocyte survival and growth. In contrast lectin-aggregated primordial follicles cultured on non-coated wells showed significantly (P < 0.05) improved oocyte survival to 50% and increased median oocyte diameter compared to non-aggregated follicles. In Experiment 3, the effect of KIT ligand (KL) at 0 ng/ml, 10 ng/ml and 100 ng/ml, on lectin-aggregated primordial follicles cultured on non-coated wells was tested. KL at 100 ng/ml significantly (P < 0.05) increased median oocyte diameter compared to non-treated controls but had no effect on oocyte survival. In addition, follicles cultured with 100 ng/ml KL expressed mRNA for AMH, a gene expressed only in granulosa cells of growing follicles. In conclusion, culture of lectin-aggregated primordial follicles supported the long-term survival and growth of oocytes from isolated sheep primordial follicles. Culture of lectin-aggregates with 100 ng/ml KL further increased oocyte growth and induced granulosa cell differentiation.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

Mouse oocyte development in vitro with various culture systems

These experiments were designed to determine whether or not hormones are required for the growth of mouse oocytes and to assess the possible role of companion granulosa cells in oocyte growth. To approach these problems, four systems for the culture of oocytes, either alone or in association with granulosa cells, were utilized: (1) isolated oocyte culture, (2) isolated oocyte-ovarian cell coculture, (3) isolated follicle culture, and (4) ovarian organ culture. Oocytes from 8-day-old B6D2F₁ mice failed to grow in isolated oocyte culture. Addition of follicle-stimulating hormone (FSH), 17β-estradiol (E₂), or serum to the medium failed to prevent oocyte degeneration or to promote oocyte growth. On the other hand, oocytes in isolated follicle culture or in organ culture grew significantly in defined medium. The results showed that oocytes grown in isolated follicle culture under defined conditions and in the absence of gonadotropins resemble oocytes grown in vivo in terms of their ultrastructural characteristics, with the exception of enlarged mitochondria. In addition, these oocytes were shown to exhibit some normal functional characteristics in terms of their increased levels of CO₂ evolution from exogenous pyruvate, and the ability of the fully grown oocytes to initiate meiotic maturation when freed from granulosa cells. It was concluded that gonadotropins are not necessary for oocyte growth and that gonadotropins are not required to potentiate the spontaneous meiotic maturation of oocytes which occurs after their isolation from granulosa cells. The results indicated that association of granulosa cells and oocytes was necessary for oocyte growth. However, isolated oocytes in coculture with ovarian cells failed to grow. Addition of FSH or E₂ to the cocultures failed to promote oocyte growth or delay oocyte degeneration. It was concluded that, under the culture conditions used, granulosa cells must be in contact with the oocyte, perhaps by means of specialized cell junctions, for oocyte growth to occur.     

Atmospheric air vs. normal middle ear gas: Effects on in vitro growth and collagen synthesis in normal middle ear fibroblasts

Summary The present study was undertaken to quantitate the effects of atmospheric air and normal middle ear gas on cultured fibroblasts obtained from normal rabbit middle ear mucosa. The cells were exposed to three different gas compositions: 7% O₂:5% CO₂:88% N₂, 21% O₂:5% CO₂:74% N₂, and 75% O₂:5% CO₂:20% N₂. The growth was monitored by measuring the total content of cell protein, the amount of DNA, and the cell division activity. The activity of the synthetic apparatus was determined by the collagen synthesis. For comparison, rabbit skin fibroblasts were grown under identical conditions. The results demonstrated significantly higher replication rate of middle ear fibroblasts at 7% oxygen than at atmospheric air whereas the collagen synthesis was significantly lower at 7%. Furthermore, the responses varied significantly between rabbit middle ear and rabbit skin fibroblasts. Thus the present study substantiates the hypothesis of an influence of atmospheric air on the middle ear mucosa which might be of importance, e.g., in relation to insertion of ventilation tubes or longstanding perforations of the tympanic membrane in otitis media.     

Development of primordial and prenatal follicles from undifferentiated somatic cells and oocytes in the hamster prenatal ovary in vitro: effect of insulin

Fetal hamster ovaries were cultured for up to 16 days in the presence or absence of various dosages of insulin to evaluate the induction of folliculogenesis in vitro. In the absence of insulin, a few primordial follicle-like structures appeared by the 4th day, and distinct primary follicles (stage 1) appeared by the 12th day of culture. The organelles in the oocytes and adjacent granulosa cells developed along with follicular growth. Moreover, gap junctions between the oocyte and somatic cell plasma membrane also developed as early as 8 days in culture. In the presence of 0.2 microg/ml insulin, primary follicles developed after 8 days, and approximately 4% secondary follicles with 2-3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 microg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.     

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1+/-0.78microm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean+/-S.E.M.) of oocytes of reorganized follicles increased (P<0.05) from 47.1+/-2.2microm to 65.3+/-2.6microm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4+/-2.0microm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7+/-1.6microm to 98.8+/-2.1microm (P<0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.     

Growth and antrum formation of bovine preantral follicles in long-term culture in vitro

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.     

Isolation and characterization of canine advanced preantral and early antral follicles

This study was designed to develop preantral follicle isolation and classification protocols for the domestic dog as a model for endangered canids. Ovary donors were grouped by age, size, breed purity, ovary weight and ovary status. Ovaries were randomly assigned to 1 of 3 digestion protocols: A) digestion and follicle isolation on the day of spaying; B) storage at 4 degrees C for 18 to 24 h prior to digestion and follicle isolation; C) digestion on the day of spaying, then incubation at 4 degrees C for 18 h prior to follicle isolation. Minced tissue was placed in a collagenase/DNase solution at 37 degrees C for 1 h. Follicles were classified by oocyte size and opaqueness and by size and appearance of the granulosa cell layers. Preantral follicles contained small, pale oocytes. Preantral follicles containing grown oocytes with dense cytoplasmic lipid were designated as advanced preantral. Only advanced preantral and early antral follicles were examined and classified further. Group 1 follicles had incomplete or absent granulosa layers, Group 2 follicles had several intact granulosa layers, while Group 3 were vesicular (early antral) follicles. Misshapen or pale grown oocytes were classified as degenerated. The percentage of intact germinal vesicles (GV) was recorded for each Group. Digestion Protocol B produced the lowest percentage of degenerated follicles (P < 0.01). Prepubertal donors had fewer (P < 0.01) follicles in each Group and more (P < 0.001) degenerated follicles than older bitches. Larger ovaries yielded the highest total number of follicles (P < 0.05). Ovary status did not affect follicle yield. Oocytes from Group 1 follicles had fewer intact GVs than those from Group 2 or Group 3 (P < 0.0001). These findings provide an opportunity for quantitative studies of the factors regulating folliculogenesis in the domestic dog as a model for endangered canids.     

Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation

Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation.     

Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles

Isolated morphologically normal bovine preantral follicles (40 to 70 microm) were cultured for 8 d in collagen gel in control medium or in 1 of 3 conditioned media from the murine granulosa cell lines GRMO1L, GRMO2 and GE2. The percentages of follicles at Day 1 that remained nomal at Day 8 were similar for follicles cultured in the conditioned and control media (84 to 90%). A significantly higher percentage of follicles cultured in each of 3 conditioned media started to grow (89%; P < 0.05) and their increase in diameter was greater than that of follicles cultured in control medium (72%; P < 0.05). The mitotic activity of the granulosa cells from follicles cultured in conditioned media was increased (P < 0.05) indicated by a higher percentage of nuclei that incorporated BrdU compared with that of follicles cultured in control medium. Follicular viability was measured by the presence of nonspecific esterase activity, active mitochondria and dead cells in cultured follicles using the fluorescent probes calcein-AM, rhodamine 123 and ethidium homodimer-1 in combination with confocal laser scanning microscopy. The percentages of follicles with esterase activity and active mitochondria present in their granulosa were similar for follicles in all groups. Culturing in GRMO2 or GE2 tended to lower the number of granulosa with dead cells. The percentage of follicles with oocytes without esterase activity and active mitochondria was lower (P < 0.05) in follicles cultured in GRMO2 or GE2 compared with those cultured in control medium. Moreover, the percentages of dead oocytes tended to be higher in follicles cultured in GRMO1L and GE2 compared with oocytes of follicles incubated in control medium. Taken together, the conditioned media stimulated follicular growth and granulosa viability as well as enhance mitotic activity of the granulosa cells. However, they negatively affected oocyte viability.     

Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro

Porcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO₂ in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs-Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN-formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle-stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.     

Birth of a Western Lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer

A 21-year-old multiparous female exhibiting 31–41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocyctes (n = 11) were placed in modified human tubal fluid (mHTF, 100 μl) medium under oil at 37°C in humidified 5% CO₂. Frozen semen, collected by voluntary ejaculation, was thawed (70°C H₂O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247–260, 1997. © 1997 Wiley-Liss, Inc.     

Effects of follicular atresia and size on the developmental competence of bovine oocytes: a study using the well-in-drop culture system

The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.     

Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P<0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO(2) in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1x ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P<0.05) in the absence of ITS+ and in the presence of 0.5x ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5x ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.