Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

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.     

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.     

Factors controlling the growth of bovine primary and preantral follicles in perifusion culture

Tissue slices taken from the cortex of bovine ovaries were perifused with medium-199 supplemented with 1) no hormones, 2) insulin (200 IU/l), 3) estradiol-17beta (E2 100 ng/ml), 4) insulin plus E2 or 5) insulin, E2 plus human chorionic gonadotropin (hCG; 0.1 IU/ml). After 0, 3, 24 and 48 h of perifusion, cortical slices were either incubated with 3H-thymidine to determine the amount of 3H-thymidine incorporated into DNA, or prepared for histology. Prior to perifusion, cortical slices incorporated 3H-thymidine at a rate of 17.5 +/- 2.5 cpm/mg/h and contained 22.2 +/- 4.4 primary and 11.3 +/- 3.9 preantral follicles/slice. 3H-thymidine incorporation remained at 0 h levels, but by 48 h of perifusion the number of primary follicles per slice was reduced to 2.5 +/- 2.2, 2.5 +/- 2.5 and 3.3 +/- 1.2 for cortical slices exposed to either no hormones, insulin or E2, respectively (P < 0.05). Exposure to either insulin and E2 or insulin, E2 plus hCG resulted in an increase in 3H-thymidine incorporation with the number of primary follicles decreasing at 24 h (P < 0.05) then increasing at 48 h of perifusion (P < 0.05). The addition of hCG increased both 3H-thymidine incorporation and the number of primary follicles present after 48 h compared to treatment with insulin and E2 alone (P < 0.05). The interstitium was well maintained if insulin was present in the medium. These results indicate that 1) insulin is essential for the maintenance of the interstitial cells, 2) a synergistic interaction between insulin and E2 stimulates primordial follicles to grow into primary follicles and 3) hCG facilitates the growth-promoting actions of insulin and E2.     

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.     

Effect of hormones and growth factors on in vitro development of sheep preantral follicles

The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 μm) and large (>250–400 μm) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T₄), FSH, LH, transforming growth factor alpha (TGF-), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonate-buffered tissue culture medium 199, supplemented with 50 μg ml⁻¹ gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T₄ + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage.     

The early stages of follicular development: activation of primordial follicles and growth of preantral follicles

Although enormous progress has been made in understanding the events and regulation of the later stages of ovarian follicular development, the early stages of development, to a large extent and particularly in large mammals, remain a mystery. Mechanisms that regulate the initiation of follicular growth (follicle activation) and the ensuing growth and differentiation of preantral follicles are of considerable interest, since their elucidation is a prerequisite to use of the primordial pool to enhance reproductive efficiency in domestic animals, humans, and endangered species. This review is an attempt to summarize the approaches that have been taken to further this goal and the results thus far of these efforts. Preantral follicular development can be divided into three stages: activation of primordial follicles, the primary to secondary follicle transition, and the development of secondary follicles to the periantral stage. The activation of primordial follicles in vitro has been achieved thus far in rodents, cattle, and primates, where it occurs spontaneously without the addition of growth factors or hormones. The ovaries of rodents are small enough to be cultured intact and, in that experimental situation, some follicles activate, while many remain in the resting pool, and the addition of specific factors can increase or decrease the number of follicles that leave the resting pool in vitro. In contrast, follicular activation in cattle and primates has been studied by culturing small pieces of the ovarian cortex, rich in primordial follicles, and the great majority of the primordial follicles activate in that situation, suggesting the importance of inhibitory factors to the normal, gradual exit of follicles from the resting pool. In cultured rodent ovaries, follicles appear to pass easily and spontaneously from the primary to the secondary stage, whereas few of the activated follicles in cultured cortical pieces from cattle or primates progress from the primary to the secondary stage. Understanding the requirements for the primary to secondary transition is critical for growing follicles activated in vitro to the late preantral and antral stages. In contrast, the requirements for the continued growth of larger preantral follicles, which can be isolated for in vitro studies, have been extensively explored in rodents and to a lesser extent in domestic species. A number of hormones and factors have been implicated and will be discussed. Taken together, the results highlight the need for a better understanding of the earliest stages of follicular development in domestic ruminants, particularly follicle activation and the primary to secondary follicle transition.     

Effects of Kit Ligand and anti-Kit antibody on growth of cultured mouse preantral follicles

Paracrine regulations between the oocyte and granulosa cells are likely to be key regulators of early folliculogenesis. Evidence obtained from genetic mutants as well as in vivo experiments suggest that Kit and Kit Ligand (KL) may regulate early follicular morphogenesis and function. In this study, we used in vitro culture of intact mouse follicles to confirm and extend these findings. Two concentrations of Kit Ligand (20 and 50 ng/ml) or an antibody blocking the Kit-Kit Ligand interactions (SC1494) were added to preantral follicles grown individually for 12 days and which were finally triggered to ovulate. Effects on follicle and oocyte survival, granulosa cell function (antrum formation, cell numbers, steroidogenesis), and oocyte function (growth, survival, nuclear and/or cytoplasmic maturation) were then analyzed. In optimal culture conditions (presence of 5% fetal calf serum), 50 ng/ml of KL significantly improved cytoplasmic maturation of the oocyte and increased follicular testosterone output, but other parameters were not altered. In serum-free culture conditions, KL was mitogenic for granulosa cells at 50 ng/ml, but could not induce antrum formation and no differences were observed between control and treated groups for steroidogenesis or oocyte growth. Blockade of Kit-Kit Ligand interactions by addition of a blocking antibody decreased oocyte survival 6-9 days after addition of the antibody, but did not upset granulosa cell proliferation. Antrum formation was, however, strongly inhibited. In addition, the blocking antibody markedly reduced aromatase activity of granulosa cells. We conclude that Kit/KL interactions are important for antrum formation and follicular steroidogenesis and regulate survival and cytoplasmic maturation of the oocyte.     

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.     

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.     

Impact of peptide growth factors on the culture of small preantral follicles of domestic cats

Small preantral follicles (40 to 90 microm) of domestic cats were cultured in the presence or absence of epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) for 5 d. The success of culture was estimated by in vitro incorporation of Brom-desoxyuridine (BrdU) into the oocytes and granulosa cells. Addition of EGF (4, 20, or 100 ng/ml) to the culture medium had no significant effect on the incidence of in vitro DNA synthesis. After supplementation with IGF-I and bFGF, BrdU-incorporation into the follicles and oocytes increased in correspondence to the concentration used, with 20 ng/ml IGF-I and 10 ng/ml bFGF giving the highest effect. In medium containing EGF, the IGF-I-induced increase in BrdU incorporation was suppressed, while the effect of bFGF was not decreased. Simultaneous addition of IGF-I and bFGF did not result in a further increase in DNA synthesis in the oocytes and granulosa cells. We conclude that bFGF mainly induces the proliferation of granulosa cells while IGF-I is involved in cellular activation of oocytes, which is modulated by EGF.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

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).     

Effects of recombinant activin A on in vitro culture of mouse preantral follicles

Activins are part of the intragonadal factors that can modulate the actions of gonadotropins and regulate cellular functions during preantral or early antral stages of folliculogenesis in vivo. In a mouse early preantral follicle culture system, activin A production was measured and recombinant bovine activin A (r-ACT A) was added (10 or 50 ng/ml) to recombinant follicle-stimulating hormone (r-FSH)–supplemented (10 or 100 mIU/ml) medium for a 12-day culture period. Specificity of activin A action was ascertained by addition of recombinant human follistatin (r-FS; 20 or 100 ng/ml). Immunoreactive activin A concentrations in mouse follicle–conditioned medium increased by a factor of 20–50, reaching concentrations from 2 to 5 ng/ml at end of culture. In the initial days of culture, additions of r-ACT A to r-FSH-supplemented medium provoked a dramatic volumetric increase and earlier attachment of the follicle. A dose of 100 ng/ml r-FS was able to block the actions of 10 ng/ml but not those caused by 50 ng/ml r-ACT A. In follicle cultures supplemented with 10 mIU/ml r-FSH, additions of r-ACT induced a dose-dependent inhibin (INH) and estradiol (E₂) increase. Basal and human chorionic gonadotropin (HCG)–induced progesterone (P) production were not influenced by r-ACT A or r-FS additions. Addition of r-ACT A decreased (P = 0.017) the intact follicle survival rate and had no influence on final oocyte diameter. In cultures supplemented by 10 mIU/ml r-FSH, additions of r-ACT A did not influence progression and resumption of meiosis I. Use of a higher r-FSH supplementation dose (100 mIU/ml) tended to affect meiosis I adversely (P = 0.052), and r-ACT A addition amplified this effect significantly (P = 0.007). These in vitro experiments demonstrate pronounced effects from r-ACT on r-FSH-mediated follicle survival, growth, and estrogen biosynthesis. Mol. Reprod. Dev. 50:294–304, 1998. © 1998 Wiley-Liss, Inc.     

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.