In vitro grown sheep preantral follicles yield oocytes with normal nuclear-epigenetic maturation

Background

Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation.

Methodology/Principal Findings

In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes.

Conclusions/Significance

In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.     

In vitro culture of buffalo (Bubalus bubalis) preantral follicles

Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin-transferrin-selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media. Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO2 incubator (38-39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 15 days. Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 microg/ml), respectively. In Experiment 1, we placed isolated preantral follicles individually or in groups of 2-4 preantral follicles in 30 or 50 microl droplets, respectively, using two culture media: washing media and washing media + ITS (1%) + FSH (0.05 IU/ml), respectively. In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: < or = 36, 37-54, 55-72, 73-90, 90-108 and > or = 109 microm. We cultured groups of 2-4 preantral follicles in washing media + ITS (1A) + FSH (0.05 IU/ml) in a CO2 incubator for 15 days. In Experiment 3, we allocated groups of 2-4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media + FSH (0.05 IU/ml), (3) washing media + ITS (17%), (4) washing media + ITS (1%) + FSH (50 IU/ml), (5) washing media + ITS (1%) + EGF (50 ng/ml), (6) washing media + ITS (1%) + FSH (0.05 IU/ml) + EGF (50 ng/ml), (7) washing media + ITS (1%) + FGF (50 ng/ml), (8) washing media + ITS (1%) + FSH (0.05 IU/ml) + FGF (50 ng/ml), (9) washing media + ITS (1%) + VIP (50 ng/ml), and (10) washing media + ITS (1%) + FSH (0.05 IU/ml) + VIP (50 ng/ml). In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly (P < 0.05) higher growth up to 40 days. In Experiment 5, we allocated groups of 2-4 preantral follicles to two treatments: (1) washing media + PMSG (50 IU/ml), and (2) washing media + ITS (1%) + PMSG (50 IU/ml) and cultured in a CO2 incubator for 15 days. The results indicated that the preantral follicles cultured in groups had a higher growth rate (P < 0.05) than those cultured as individuals. ITS, FSH, PMSG and growth factors significantly (P < 0.05) promoted the growth of the preantral follicles. Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation. The growth rate of preantral follicles was lower in buffalo than in cattle.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

水牛腔前卵泡的分离方法

为确定水牛腔前卵泡的有效分离方法 ,作者取用来自屠宰场 31头水牛的 6 1个卵巢 ,先用剪刀去掉髓质部 ,然后分别用梳刮法、剪碎法和显微分离法分离回收腔前卵泡。结果发现 ,使用梳刮法时 ,平均每个水牛卵巢回收所得的腔前卵泡数 (15 2 35± 4 4 81)明显高于剪碎法 (32 6 2± 14 81)和显微分离法 (8 95± 3 4 4 ) ,且其平均每个卵巢的处理时间 (39 0 5± 4 2 7min)明显少于剪碎法 (4 6 4 3± 4 15min)和显微分离法 (4 4 5 5± 7 82min)。梳刮法分离所得的腔前卵泡中 ,以原始卵泡最多 (占 4 1 2 5 % ) ,其次为初级卵泡 (占 38 79% ) ,而次级卵泡最少 (仅占 19 95 % )。用梳刮法获得的腔前卵泡在体外培养 72h后 ,存活率为 5 6 38% ,与剪碎法(4 8 91% )及显微分离法 (5 9 34% )没有显著差异。用梳刮法处理 10头黄牛的 2 0个卵巢 ,平均每个卵巢可分离获得 1195 2 0± 6 85 0 0个腔前卵泡 ,明显多于水牛的 15 2 35± 4 4 81个腔前卵泡。由此可见 ,梳刮法是水牛腔前卵泡的有效分离方法     

Development of morulae from the oocytes of cultured sheep preantral follicles

Sheep preantral follicles (PFs) measuring 250-400 μm in diameter were cultured for six days in serum-free media supplemented differently with growth factors and hormones. Subsequently, oocytes from the cultured follicles were subjected to an additional 24 h of in vitro maturation (IVM) followed by in vitro fertilization (IVF) and embryo culture for 6 days. Five different experiments were conducted. In the first experiment individual concentrations of Insulin-Transferrin-Selenite (ITS), Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta (TGF-β), Insulin (INS), and Growth hormone (GH) that supported the best in vitro development of the PFs were determined. The influence of different combinations of the above hormones and growth factors at their best concentrations as determined in the first experiment was investigated in the second experiment. In the third experiment the best combinations of the growth factors and hormones obtained in the second experiment were additionally supplemented with Thyroxin (T4) and follicle stimulating hormone (FSH) and the influence on in vitro development of the PFs was studied. In the fourth experiment, two methods of culturing PFs—micro drops and agar gel embedding—were compared. In the fifth experiment oocytes from cultured PFs were subjected to IVF and in vitro development of the resulting embryos was followed to the blastocyst stage.Based on the proportion of the PFs exhibiting growth, mean increase in diameter, proportions of PFs developing antrum, ovulations in vitro and oocytes maturing to M-II stage, 1% ITS, 10 ng/mL each of IGF-I, and Insulin and 1 mIU/mL of GH were found to support the best development of sheep PFs. However, the oocytes from PFs cultured in any concentration of TGF-β failed to mature to M-II stage. Similarly, among the combinations studied, IGF-I+GH was found to be the best. In combination with T4 and FSH, IGF-I+GH supported the best development of the PFs. Culture of PFs in micro drops or agar gel supported similarly high development. In vitro fertilization of the oocytes from the cultured sheep PFs resulted in the embryos developing to the morula stage for the first time.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.     

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.     

In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage.     

In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

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.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

Recovery of large preantral follicles from buffalo ovary: effect of season and corpus luteum

Preantral follicle can be considered as an alternative source of oocyte for in vitro production of embryos. The objective of the present study was to standardize a procedure for the isolation of large preantral follicles (>150-500 microm) from buffalo ovaries and to determine the effect of season and the presence of corpus luteum on the recovery rate of the large preantral follicles. A combined enzymatic cum mechanical approach was adopted to recover the large preantral follicles. In the first experiment, the ovarian cortical pieces were suspended in trypsin (1000-1500 BAEE units for milligrams of solid) and incubated at various temperatures for different periods, i.e. (1) trypsin (1%), 37 degrees C for 10 min; (2) trypsin (1%), 37 degrees C for 10 min + 4 degrees C for 3 h; (3) trypsin (0.5%), 37 degrees C for 20 min; (4) trypsin (0.25%), 37 degrees C for 20 min. Although there was no significant difference (P>0.05) among the different protocols, the first protocol yielded more follicles (3.2, 2.6, 1.8 and 1.5 per ovary, respectively). Hence, the first protocol was selected and used in the second and third experiments. In the second experiment, the effect of season, i.e. peak breeding season (October-March) versus low breeding season (April-September) was evaluated on the recovery rate of the large preantral follicles. The recovery rate of large preantral follicles from the ovaries during the peak breeding season was significantly (P<0.05) greater (9.92+/-0.85 per ovary) than that of the low breeding season (4.95+/-0.27 per ovary). In the third experiment, effect of the presence of corpus luteum on the recovery rate of large preantral follicles was studied. There was a significantly (P<0.05) higher yield of large preantral follicles from the ovaries with corpus luteum (8.05+/-0.88 per ovary) than for the ovaries without corpus luteum (4.57+/-0.43 per ovary). This study confirms that the large preantral follicles can be isolated from buffalo ovaries using a combination of enzymatic cum mechanical methods and that more large preantral follicles can be recovered during the peak breeding season and from the ovaries having corpus luteum.     

In vitro development of caprine ovarian preantral follicles

The in vitro growth and developmental pattern of caprine preantral follicles cultured in agar gel was observed. Preantral follicles 50 to 150 microm in diameter were isolated from prepuberal goat ovaries by treatment with collagenase and DNase. The isolated preantral follicles were cultured in agar gel for up to 14 days. A group of 10 follicles in different developmental stages was cultured in a culture well coated with 0.6% agar gel and filled with DMEM medium supplemented with FCS (10%), hypoxanthine (2 mmol/mL), dbcAMP (2 mmol/mL), FSH (100 ng/mL), insulin-transferrin-selenium (ITS) (50 ng/mL), IGF-1 (50 ng/mL), hydrocortisone (40 ng/mL) and antibiotics. Follicle viability was determined under an inverted phase-contrast microscope according to morphological and histological criteria, and follicle growth was assessed by their size and appearance. The results showed that the three-dimensional structures and forms of follicles were basically maintained intact during culture. Primary follicles developed into secondary follicles and a few of them into antral follicles. A large portion of secondary follicles entered the antral stage, and oocytes also acquired growth. The formation of theca lamina and zona pellucida was observed. The survival capacity of secondary follicles was greater than primary follicles. The survival rates for primary and secondary follicles were 11.36% (5/44) and 71.16% (53/74), respectively. During in vitro development the follicles demonstrated dominance. This experiment revealed the preliminary characteristics of the in vitro development of caprine preantral follicles.     

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.     

Ultrastructural and morphometric characterization of buffalo (Bubalus bubalis) ovarian preantral follicles

The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 microm and the oocyte diameter is 24.9 microm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 microm) consist of an oocyte, with a medium diameter of 26.9 microm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 microm, and the oocyte has a mean diameter of 29.4 microm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell-oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species.     

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.     

哺乳动物腔前卵泡体外培养研究进展

哺乳动物卵巢中含有数以万计的腔前卵泡（preantral follicles）,仅不足1％能够发育至预排卵卵泡。建立哺乳动物腔前卵泡有效分离及体外培养技术体系,能够大量利用腔前卵泡,增加体外成熟卵母细胞数量,对哺乳动物胚胎体外生产、克隆及转基因动物生产等胚胎工程技术的研究与应用,以及在体外条件下揭示哺乳动物卵泡发育机理,都具有重要的理论意义和实用价值。鉴于卵巢质地与卵泡划、发生周期的固有差异,不同物种腔前卵泡分离方法与培养方式亦有所不同。同时,培养基类型、卵泡问相互作用、促性腺激素与细胞因子等因素均会对卵泡的体外发育产生影响。系统阐述了腔前卵泡的分离方法、培养方式以及相关因素对卵泡发育的影响,期望为从事相关研究的学者提供参考。