In vitro maturation of bitch oocytes from advanced preantral follicles in synthetic oviduct fluid medium: serum is not essential

The ability of oocytes from preantral follicles to mature in vitro was assessed using a synthetic oviduct fluid (SOF) medium. Advanced preantral follicles (approximately 210 microm diameter) were isolated from the ovaries of domestic bitches and assigned to one of four treatment groups: (1) SOF (n = 230); (2) SOF + 3 mg/ml bovine serum albumin (+BSA, n = 220); (3) SOF + 20% fetal bovine serum (+FBS, n = 227); or (4) SOF + 3 mg/ml BSA + 20% FBS (+BSA+FBS, n = 232), then cultured for up to 72 h. A group of control follicles was not cultured (n = 103). The percentages of oocytes reaching metaphase I to metaphase II stages (MI to MII) did not differ between treatments at each culture period. Within treatments, the percentages of oocytes at MI to MII stages did not differ with duration of culture. However, when compared to the control group (0.97%) the percentages of oocytes at MI to MII increased (P < 0.05) in the SOF group after 48 h (10.0%) and 72 h (12.2%) of culture. In the +BSA (10.1%) and +FBS (9.7%) groups, the percentages of oocytes at MI to MII increased (P < 0.05) above control values only after 72 h of culture. The percentage of oocytes at MI to MII did not significantly increase in the +BSA+FBS group (3.9,6.6 and 7.6% at 24,48 and 72 h of culture, respectively) compared to the control group. These results indicate that under the described conditions supplementation of culture medium with BSA or FBS is not essential, and the simple medium SOF can support nuclear maturation of a small proportion of bitch oocytes in vitro.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Effects of gonadotrophins, growth hormone, and activin A on enzymatically isolated follicle growth, oocyte chromatin organization, and steroid secretion

So far, standard follicle culture systems can produce blastocyst from less than 40% of the in vitro matured oocytes compared to over 70% in the in vivo counterpart. Because the capacity for embryonic development is strictly associated with the terminal stage of oocyte growth, the nuclear maturity status of the in vitro grown oocyte was the subject of this study. Mouse early preantral follicles (100-130 microm) and early antral follicles (170-200 microm) isolated enzymatically were cultured for 12 and 4 days, respectively, in a collagen-free dish. The serum-based media were supplemented with either 100 mIU/ml FSH (FSH only); 100 mIU/ml FSH + 10 mIU/ml LH (FSH-LH); 100 mIU/ml FSH + 1 mIU/ml GH (FSH-GH) or 100 mIU/ml FSH + 100 ng/ml activin A (FSH-AA). Follicle survival was highest in follicle stimulating hormone (FSH)-AA group in both cultured preantral (91.8%) and antral follicles (82.7%). Survival rates in the other groups ranged between 48% (FSH only, preantral follicle culture) and 78.7% (FSH only, antral follicle culture). Estradiol and progesterone were undetectable in medium lacking gonadotrophins while AA supplementation in synergy with FSH caused increased estradiol secretion and a simultaneously lowered progesterone secretion. Chromatin configuration of oocytes from surviving follicles at the end of culture revealed that there were twice more developmentally incompetent non-surrounded nucleolus (NSN) oocytes (>65%) than the competent surrounded nucleolus (SN) oocytes (<34%). We conclude that the present standard follicle culture system does not produce optimum proportion of developmentally competent oocytes.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Effects of culture medium,serum type,and various concentrations of follicle-stimulating hormone on porcine preantral follicular development and antrum formation in vitro

Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.     

Protein kinase C activity regulates the onset of anaphase I in mouse oocytes

The metaphase-to-anaphase I transition is a key step in the completion of meiosis I. In mouse oocytes, competence to exit metaphase I (MI) is developmentally regulated and typically not acquired until the preovulatory stage. The possible role of protein kinase C (PKC) in regulating this critical transition was assessed in both normal oocytes isolated from small antral follicles (18-day-old B6SJLF1 mice), which have not yet developed the capacity to progress to metaphase II (MII), and also oocytes defective in their ability to exit MI despite development to the preovulatory stage (24-day-old CX8 recombinant inbred strains). In both systems, transient suppression of endogenous PKC activity by treatment with a PKC-specific inhibitor, bisindolylmaleimide I (BIM), promoted the onset of anaphase I in a dose-dependent manner, while activation of PKC with the phorbol ester TPA blocked progression to MII. Following a 2-h incubation with BIM, the majority of oocytes progressed to, and arrested at, MII. The resulting MII oocytes were fertilizable in vitro, showing similar cleavage and blastocyst development rates between BIM treated and untreated controls. Transferred embryos resulted in the development of pups to term in both groups. These data demonstrate that PKC plays an important role in regulating the onset of anaphase I in mouse oocytes. Moreover, it is concluded that oocytes isolated from small antral follicles become blocked at MI due to a PKC-mediated signal, suggesting that acquisition of competence to complete meiosis I involves, in part, the control of PKC activity. Similarly, failure to regulate PKC activity at the preovulatory stage likely promotes arrest at MI.     

Follicular interactions affect the in vitro development of isolated goat preantral follicles

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

In vitro development of sheep preantral follicles

Preantral ovarian follicles isolated from prepubertal sheep ovaries were individually cultured for 6 days in the presence of increasing doses of FSH (ranging from 0.01 to 1 microg/ml) and under two different oxygen concentrations, 20% and 5% O2. Follicle development was evaluated on the basis of antral cavity formation as well as the presence of healthy cumulus oocyte complexes. Follicle growth was enhanced by FSH addition to culture medium, while the use of a low oxygen concentration slightly stimulated this process. However, when follicles were cultured in the presence of high doses of FSH (1 microgram/ml) and under low oxygen concentration, a high proportion of them showed the presence of an antral cavity and of a healthy cumulus-oocyte complex. In addition, under this specific culture condition sheep preantral follicles released higher levels of estradiol as compared to those secreted at lower FSH concentrations or under 20% O2. When the meiotic competence of oocytes derived from follicles cultured at 1 microgram/ml FSH was assessed, no significant difference was recorded between the two oxygen groups. These results show that the culture conditions here identified are beneficial to in vitro growth and differentiation of sheep preantral follicles.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

Insulin signaling in mouse oocytes

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-beta. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 microg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 microg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.     

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation

The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system

In vivo studies on X-irradiated mice have shown that structural chromosome aberrations can be induced in female germ cells and that the radiation-induced chromosomal damage strongly depends on the stage of maturation reached by the oocytes at the time of irradiation. In the present study, the sensitivity of oocytes to induction of chromosome damage by radiation was evaluated at two different stages, by use of a recently developed method of in vitro culture covering a crucial period of follicle/oocyte growth and maturation. A key feature of this system is that growth and development of all follicles is perfectly synchronized, due to the selection of a narrow class of follicles in the start-off culture. This allows irradiation of well-characterized and homogenous populations of follicles, in contrast to the situation prevailing in vivo. Follicles were X-irradiated with either 2 or 4 Gy, on day 0 of culture (early preantral follicles with one to two cell layers) or on day 12, 3h after hormonal stimulation of ovulation (antral Graafian follicles). Ovulated oocytes, blocked in metaphase I (MI) by colchicine, were fixed 16 h after hormonal stimulation and analyzed for chromosome aberrations. The results confirm the high radiosensitivity of oocytes at 2 weeks prior to ovulation and the even higher radiosensitivity of those irradiated a few hours before ovulation, underlining the suitability of the in vitro system for further studies on the genetic effects of ionising radiation in female mammals.     

Factors affecting follicular population, oocyte yield and quality in camels (Camelus dromedarius) ovary with special reference to maturation time in vitro

Three experiments were conducted to study a series of factors affecting in vitro reproductive parameters in camels. In Experiment 1, the effect of season and presence of a corpus luteum (CL) on ovarian follicular populations, oocyte yield and quality was studied using a total of 252 and 208 ovaries collected during the breeding and non-breeding season, respectively. Small, medium, large and the total number of ovarian follicles, oocyte yield and quality were measured. In Experiment 2, the effect of methods of oocyte retrieval and needle gauge on oocyte yield and quality was evaluated with oocytes recovered using slicing and aspiration with 18-, 19- or 20-gauge needle. Oocytes were evaluated microscopically and classified into three categories. The objective of Experiment 3 was to identify the optimum time for oocyte maturation in the dromedary camel. Oocytes were cultured in CR1aa medium at 38.5 degrees C under 5% CO(2) for 24, 32, 36, 48 and 72h. Maturation was calculated as the percentage of cumulus expansion and oocytes reaching metaphase II (MII). The number of small, medium, large and the total number of ovarian follicles were higher (P<0.01) during the breeding than non-breeding season. The recovery of total number of oocytes and Category I oocytes were also greater (P<0.01) during the breeding season. Ovaries without a CL possessed significantly (P<0.01) more ovarian follicles and more (P<0.05) small and large follicles. The total number of oocytes and Category I oocytes were also greater (P<0.01) in ovaries without CL. Slicing of camel ovaries increased (P<0.01) the yield of oocytes as compared to aspiration. The aspiration of follicles using a 20-gauge needle had greater yields of the total number of oocytes and Category I oocytes than when using 19- (P<0.05) and 18-gauge needle (P<0.01). The culture of camel oocytes for 36h produced higher (P<0.01) percentages of cumulus expansion and oocytes at MII. Increasing culture times up to 48 or 72h increased (P<0.01) the percentage of degenerated oocytes.In conclusion, the growth and development of ovarian follicles in the camel as well as yields of Category I oocyte were greater during the breeding season. Slicing or aspirations using a 20-gauge needle yielded greater numbers of total and Category I oocytes. Finally, maturation of oocytes in CR1aa medium for 36h produced higher percentages of cumulus expansion and oocytes at MII stage.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.