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.     

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 growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue.

A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.     

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 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 DNA synthesis by isolated preantral to preovulatory follicles from the cyclic mouse

In recent studies, we have shown that the smallest preantral follicles in the cyclic hamster increase DNA synthesis in the periovulatory period in response to surge levels of FSH. The current investigation was designed to determine whether the same phenomenon occurs in the cyclic mouse. Intact mouse follicles were isolated with watchmaker forceps (stages 4-6) or by enzymatic digestion (stages 1-4) at 0900 h and 1500 h on each day of the 5-day estrous cycle. The isolated follicles were classified into 6 stages: stages 1 and 2: follicles with 1 and 2 layers of granulosa cells; stage 3: follicles with 3 or more layers of granulosa cells and formation of theca; stages 4-6: incipient, small, and preovulatory antral follicles. The follicles at each stage were incubated for 3 h with [3H]thymidine. DNA content in stages 1-4 of follicles remained unchanged during the estrous cycle; for stages 5 and 6, DNA content was higher on the afternoon of proestrus than on other days of the cycle. Incorporation of [3H]thymidine for stages 1-3 (preantral follicles) started to increase at 1500 h of proestrus and peaked at 0900 h on estrus, whereas for stages 4-6, DNA synthesis peaked on proestrus (1500 h) and then fell by the morning of estrus. Thus, the rate of DNA replication in preantral and antral mouse follicles were different. Similarities and differences in folliculogenesis between mouse and hamster are discussed. These results suggest that DNA synthesis and the growth of all stages of follicles in the cyclic mouse may be associated with changing levels of periovulatory gonadotropins.     

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.     

Characterization of in vitro growth of bovine preantral ovarian follicles: A preliminary study

Preantral follicles were mechanically extracted from bovine ovaries collected at slaughter. On average 90 early growing follicles were collected per ovary. The follicles were cultured for 7 days in Dulbecco's modified Eagle medium F-12 nutrient mixture + 10% fetal calf serum + 10% newborn calf serum. The individual follicular diameter and the follicular DNA content were recorded at the start and the end of culture. The follicular DNA content was estimated by a microfluorometric method using the fluorochrome, 4'-6 diamidino-2-phenylindol-2HCl (DAPI). After culture, 86% of the follicles looked morphologically normal. Of the surviving follicles, 73% showed an increase in diameter varying between 5 and 30 mum, and the follicular DNA content increased from 1 to 72% under the same culture conditions. These results indicate that bovine preantral follicles can survive in vitro and even grow in a single medium.     

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.     

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.     

Xenobiotic effects on ovarian preantral follicles

Women are born with a finite population of ovarian follicles, which are slowly depleted during their reproductive years until reproductive failure (menopause) occurs. The rate of loss of primordial follicles is determined by genetic and environmental influences, but certain toxic exposures can accelerate this process. Ionizing radiation reduces preantral follicle numbers in rodents and humans in a dose-dependent manner. Cigarette smoking is linked to menopause occurring 1-4 yr earlier than with nonsmokers, and components of smoke, polycyclic aromatic hydrocarbons, can cause follicle depletion in rodents or in ovaries in vitro. Chemotherapeutic agents, such as alkylating drugs and cisplatin, also cause loss of preantral ovarian follicles. Effects depend on dose, type, and reactivity of the drug, and the age of the individual. Evidence suggests DNA damage may underlie follicle loss induced by one common alkylating drug, cyclophosphamide. Occupational exposures have also been linked to ovarian damage. In an industrial setting, 2-bromopropane caused infertility in men and women, and it can induce ovarian follicle depletion in rats. Solvents, such as butadiene, 4-vinylcyclohexene, and their diepoxides, can also cause specific preantral follicle depletion. The mechanism(s) underlying effects of the latter compound may involve alterations in apoptosis, survival factors such as KIT/Kit Ligand, and/or the cellular signaling that maintains primordial follicle dormancy. Estrogenic endocrine disruptors may alter follicle formation/development and impair fertility or normal development of offspring. Thus, specific exposures are known or suspected of detrimentally impacting preantral ovarian follicles, leading to early ovarian failure.     

Histological and ultrastructural analysis of cryopreserved sheep preantral follicles

The aim of this study was to verify the histological and ultrastructural characteristics of sheep preantral follicles after exposure of ovarian tissue to cryopreservation in glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) in order to determine the optimum method to store sheep ovarian tissue for later experimental or clinical use. Each ovarian pair from five mixed-breed ewes was divided into 17 fragments. One (control) fragment was immediately fixed for routine histological and ultrastructural studies and the remaining (test) fragments were randomly distributed in cryotubes, equilibrated at 20 degrees C/20 min in 1.8 mL of minimal essential medium (MEM) containing 1.5 or 3 M GLY, EG, PROH or DMSO and then either fixed for morphological studies to determine their possible toxic effect or frozen/thawed and then fixed to test the effect of cryopreservation on preantral follicles. Histological analysis showed that, compared to control fragments, all cryoprotectants at both concentrations significantly reduced the percentage of normal preantral follicles in ovarian fragments prior to or after cryopreservation. PROH 3.0 M appeared to exert a more toxic effect (P<0.05) than the other cryoprotectants in noncryopreserved tissues. After freezing/thawing, the highest (P<0.05) percentages of lightmicroscopical normal preantral follicles were observed in ovarian fragments cryopreserved in EG (1.5 and 3 M) or DMSO (1.5 M). However, transmission electronic microscopical (TEM) examination showed that only the DMSO-cryopreserved preantral follicles had normal ultrastructure. The data suggest that sheep preantral follicles should be cryopreserved with 1.5 M DMSO for later clinical or experimental application.     

Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro.

The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) and a simple combination of growth factors is reported. Preantral follicles, 150 microns in diameter, with thecal cells attached were isolated mechanically. After 6-7 days on a Millicell membrane, a high proportion of the preantral follicles cultured individually with hFSH grew to morphologically normal large antral follicles (400-500 microns in diameter) with high oestradiol secretion. Without hFSH, the follicles grew to approximately 275 microns diameter in 6 days, but did not form antra or secrete oestradiol. The growth trajectory (overall pattern of growth formed by daily measurements of diameter) of each follicle was recorded and used as a measurement of response to experimental variation of culture conditions. The rapidly growing follicles were morphologically normal, but those that grew more slowly showed some abnormality or atresia and secreted less oestradiol. Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose-response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12.5-100 miu hFSH ml-1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml-1).     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

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.     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

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.     

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.     

人腔前卵泡分离及培养

采用机械吹打、胶原酶消化、镜下显微解剖及胶原酶消化加镜下显微解剖4种卵泡提取方式并相互对比.将分离得到的腔前卵泡在含FSH0.5IU/mL、1.0IU/mL和2.0IU/mL的培养液中培养,检测其E2分泌量.与机械吹打、胶原酶消化相比,胶原酶消化加镜下显微解剖法不仅提取卵泡多(P＜0.01),而且可以得到原始、初级、次级各级卵泡.但操作时间较长(P＜0.01).得到的腔前卵泡在FSH为0.5IU/mL、1.0IU/mL和2.01IU/mL的培养液中培养,所分泌的E2分别为10.86pg±4.11pg、31.55pg±9.34pg和43.82pg±18.76pg,较之对照组的4.99pg±2.09pg有显著性差异(P＜0.01),E2的分泌量与FSH浓度呈剂量依赖性关系.FSH有促进腔前卵泡分泌E2的作用.     

The regulation of apoptosis in preantral ovarian follicles

Less than 1% of ovarian follicles ever mature to ovulation. The remainder undergo atretic degeneration via apoptosis during development. Though the regulation of antral and preovulatory survival has been studied for many years, very little is known about the regulation of survival and development of preantral follicles. This review discusses recent findings regarding preantral follicle development with emphasis on the regulation of preantral follicle apoptosis.