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.     

Activin promotes oocyte development in ovine preantral follicles <Emphasis Type="Italic">in vitro</Emphasis>

Activins have been implicated as important regulating factors for many reproductive processes. The aim of this study was to determine the effect of activin A on the development of ovine preantral follicles in vitro. Mechanically isolated preantral follicles (161 ± 2 microm) were cultured for 6 days in the presence of human recombinant activin A (0, 10 and 100 ng/ml). Half of the medium was replaced every second day and follicle diameters were measured. Conditioned medium was subsequently analysed for oestradiol content using a delayed enhancement lanthanide fluorometric immunoassay (DELFIA). At the end of the culture period, follicles were fixed and processed for histology, after which oocyte diameter and granulosa cell death were measured. There was significant follicle growth over 6 days in all groups (p < 0.001). Activin, at both concentrations, increased follicle growth over control levels by Day 6 (p < 0.05). Oocyte diameters were also significantly increased by Day 6 of culture in all groups (p < 0.05), with 100 ng/ml activin increasing oocyte diameter over control levels (p < 0.05). Activin, at both concentrations, increased oestradiol production on Day 2 of culture, but this increase was not sustained during the culture period. Moreover, activin did not have any effect on antrum formation or follicle survival. In conclusion, activin promoted ovine preantral follicle and oocyte growth in vitro, but did not accelerate follicle differentiation over a six-day culture period. These results support a paracrine role for activin A during early oocyte and follicular development.     

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.     

Steady‐state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro

The aims of this study were to investigate steady‐state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT‐PCR was used to analyze caprine steady‐state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1–3 mm) and large (3–6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM⁺) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT‐PCR demonstrated an increase in steady‐state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady‐state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM⁺ after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth. Mol. Reprod. Dev. 77: 231–240, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro

The effects of recombinant human activin A on the development of bovine one-cell embryos matured and fertilized in vitro were investigated. In experiment 1, one-cell embryos were cultured in a chemically-defined medium, of modified synthetic oviduct fluid supplemented with 1 mg/ml polyvinyl alcohol (mSOF-PVA), containing different concentrations of activin (0, 0.1, 1, 10, and 100 ng/ml) until 240 hr after in vitro fertilization. The addition of -1 ng/m activin to mSOF-PVA improved development to the blastocyst stage (14.5–17.1%), compared with no addition of activin (5.6%). However, there was no significant difference in hatching rate of embryos among treatments. In experiments 2 and 3, the embryos were also cultured in MSOF-PVA at various periods of exposure to 10 ng/ml activin to evaluate (development to the morula and blastocyst stages, respectively. The proportion of morulae was significantly higher in culture with activin at 20–120 hr postinsemination (37.2%) than with control (25.7%). Total number of cells in morulae at 120 hr postinsemination significantly increased by the addition of activin at 20–72 hr (26.1 cells) and 20–120 hr (24.2 cells) postinsemination, compared with control (20.1 cells). When activin was added to the medium during 20–120 hr and 20–192 hr postinsemination, the percentages of blastocysts (18.0% and 18.7%, respectively) were significantly higher than in the control (9.6%). However, the total number of cells in blastocysts was not significantly different. These results demonstrate that activin stimulates the development of bovine one-cell embryos to the morula and blastocyst stages in vitro. © 1996 Wiley-Liss, Inc.     

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

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

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.     

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.     

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.     

BMPRIB and BMPRII mRNA expression levels in goat ovarian follicles and the in vitro effects of BMP-15 on preantral follicle development

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

How the concentration of insulin affects the development of preantral follicles in goats

This study investigated the effect of adding different insulin concentrations to the culture medium for goat preantral follicle development in vitro. The ovarian fragments were immediately fixed or cultured for 7 days in MEM with insulin (0, 5, 10 ng/ml and 5 or 10 μg/ml). The results showed that, after 7 days of culture, insulin at 10 ng/ml was the best concentration to preserve follicular viability and ultrastructure, resulting in the highest rates of normal follicles. After 7 days, only treatments with 10 ng/ml and 5 μg/ml of insulin increased follicular activation when compared to other concentrations. Regarding follicular and oocyte growth, the presence of 10 ng/ml of insulin promoted a larger diameter than other treatments. In conclusion, this study shows that addition of 10 ng/ml of insulin to the culture medium improved the survival and stimulated growth of goat preantral follicles.     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.