Interactions of indole acetic acid with EGF and FSH in the culture of ovine preantral follicles

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. The objective of this study was to evaluate the effects of adding indole acetic acid (IAA), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) to the media for in vitro culture of ovine ovarian fragments and determine their effects on growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2 or 6 days in culture plates with: minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, bovine serum albumine and antibiotics (MEM+); MEM+ plus IAA (40 ng/mL); MEM+ plus EGF (100 ng/mL); MEM+ plus FSH (100 ng/mL); MEM+ plus IAA+EGF; MEM+ plus IAA+FSH; MEM+ plus EGF+FSH; or MEM+ plus IAA+EGF+FSH. After 2 or 6 days of culture in each treatment, the pieces of ovarian cortex were fixed in Bouin for histological examination. Follicles were classified as primordial or developing (primary and secondary) follicles. Compared to the control, in all media tested, the percentages of primordial follicles were reduced (P<0.05) and the percentages of developing follicles were increased (P<0.05) after 2 or 6 days of culture. Furthermore, culture of ovarian cortex for 6 days reduced the percentages of healthy, viable follicles when compared with the control (P<0.05), except for cultures supplemented with IAA+EGF and EGF+FSH. In conclusion, the addition of IAA and EGF or EGF and FSH to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture.     

Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue

Factors that control the onset of folliculogenesis are critical to female gamete production, but poorly understood. The aim of the present study was to investigate the effects of FSH and EGF on the activation and growth of goat primordial follicles in vitro. To this end, pieces of goat ovarian cortex were cultured in vitro for 1, 3 or 5 days, at 39 degrees C in an atmosphere containing 5% CO(2), in minimum essential medium supplemented with insulin, transferrin, selenium, pyruvate, glutamine, hypoxanthine, BSA, penicillin, streptomycin and fungizone and with or without FSH (100 ng/ml) and/or EGF (100 ng/ml). At the end of the culture periods, the relative proportions of primordial, intermediate, primary and secondary follicles were calculated and compared with those in non-cultured tissue. In addition, mitotic activity of granulosa cells was studied by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In brief, it was found that goat primordial follicles activate spontaneously during culture in vitro and, while neither FSH nor EGF affected the proportion of primordial follicles that entered the growth phase, both stimulated an increase in oocyte and follicle diameter, especially in intermediate and primary follicles cultured for 5 days. On the other hand, there was no significant effect of culture or either growth factor on the proportion of PCNA-stained growing follicles. Contrary to expectations, neither FSH nor EGF affected follicle viability or integrity during culture, since the percentages of intact follicles did not differ between control, FSH and/or EGF containing medium. In conclusion, this study demonstrated that goat primordial follicles activate spontaneously in vitro, and that both FSH and EGF stimulate an increase in follicle size by promoting oocyte growth.     

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM - control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.     

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.     

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

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

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.     

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

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

Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.     

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.     

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.     

Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH)

The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 μm) were isolated by microdissection and cultured for 18 d in supplemented α-Minimum Essential Medium (α-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean ± SEM) 298.96 ± 7.02, 286.00 ± 5.87, and 275.39 ± 174 6.55 um, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 ± 14.08 μm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (μm/d) of follicles in the FSHSeq treatment (6.47 ± 0.55) was significantly faster than for both the control (3.67 ± 0.32) and FSH100 (4.47 ± 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.     

Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis

Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles. The aim of the present study was to examine the in vitro effect of a GnRH-a (leuprolide acetate, LA) and its interactions with FSH, dibutyryl cAMP, and growth factors (IGF-I, EGF, and FGF) on follicular apoptosis in early antral ovarian follicles obtained from prepubertal DES- treated rats. Follicles cultured 24 hr in the absence of hormones showed spontaneous onset of apoptotic DNA fragmentation. The presence of FSH suppressed the spontaneous onset of apoptotic DNA fragmentation (75-85%). Quantitative estimation of DNA cleavage from ovarian follicles revealed no significant changes in DNA fragmentation after in vitro LA treatment (1-100 ng/ml). However, coincubation with LA interfered partially with the effects of FSH on apoptosis suppression. This apoptosis suppression was also obtained by treatment with dibutyryl cAMP (80%), and was partially prevented by the presence of LA in the cultures. Follicles were cultured 24 hr with FGF, EGF, or IGF-I, and these factors suppressed DNA fragmentation (70, 60, and 70% respectively), while the presence of LA (100 ng/ml) in the culture medium prevented this effect. In conclusion, we show that the rescue from apoptotic DNA fragmentation produced in early antral follicles by FSH, cAMP, and growth factors, is prevented by coincubation with LA. This GnRH analog would thus interfere in the pathway of FSH, cAMP and/or growth factors by an as yet unknown mechanism.     

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.     

Role and gonadotrophic regulation of X-linked inhibitor of apoptosis protein expression during rat ovarian follicular development in vitro

Although FSH up-regulates follicular cell X-linked inhibitor of apoptosis protein (XIAP) expression and suppresses apoptosis in vivo, if these events are coincidental or causally related remains to be investigated. The present study examined the role and gonadotrophic regulation of XIAP expression during follicular development in vitro. Follicles (160-210 microm) cultured for 0-6 days with FSH (100 ng/ml) showed significant growth, as evidenced by increases in follicular size, cell number, and DNA contents. Follicular XIAP content was low in the absence of FSH but was increased by the addition of gonadotropin. Apoptosis was evident in follicles cultured without FSH but was suppressed in the presence of gonadotropin. At low FSH concentration (5 ng/ml), adenoviral XIAP sense cDNA expression increased XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth. Infection of the FSH-stimulated follicles with XIAP antisense elicited opposite responses. In primary granulosa cell cultures, FSH significantly increased XIAP content, inhibited apoptosis, and decreased cell number, a response potentiated by XIAP sense expression. In conclusion, the present studies demonstrated, to our knowledge for the first time, that XIAP plays an important role in the regulation of ovarian follicular development. In addition, a follicle culture system coupled to an adenoviral gene-manipulation procedure has been established and may prove to be a useful approach in assessing the role of specific genes in follicular development and atresia.     

Luteinizing hormone has a stage-limited effect on preantral follicle development in vitro

Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 micrometer in diameter (2-3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1-2 granulosa cell layers, 85-110 micrometer in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 micrometer in diameter until the follicles had reached 150 micrometer in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.     

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.