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.     

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.     

The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d.     

Circulating gonadotropins levels and contribution of different large antral follicles to isofolliculia in sheep

The aim of this study was first to search for isofolliculia in right and left ovaries during postnatal development of Ouled Djellel ewe lambs, a non-seasonal breed of sheep. In addition, the contribution of different sizes of large antral follicles to this phenomenon was studied, and finally the variations in both plasma FSH and LH levels during this period of life were determined. Plasma was collected from groups of four ewe lambs at 0 (<24 h), 1 week, and every 2 weeks from 4 to 14 weeks of age. Thereafter, each group was slaughtered, right and left ovaries recovered, weighed and their length and width measured. One ovary was fixed in Bouin–Holland's solution and prepared for histological study. The other one was immediately frozen and cut in a cryostat and prepared for histochemical study. This latter method was used to detect the activity of alkaline phosphatase, lactate dehydrogenase, NADH₂-tetrasolium reductase, and glucose-6-phosphate dehydrogenase enzymes. Number and size of antral follicles and their contribution to isofolliculia were determined from ovarian sections of both studies. Isofolliculia was seen in right and left ovaries of Ouled Djellel ewe lambs at 4, 6, 8, and 10 weeks of age. This phenomenon was characterized by the presence of large antral follicles almost equal in size and total enzymatic inactivity in the interstitium. Weight and dimensions of right and left ovaries increased rapidly from birth to 4 weeks of age, and then rose gradually to week 8 and then rising again to week 10, followed by a decline at 12 and 14 weeks of age. All large antral follicles contributed to isofolliculia in right and left ovaries but, the percentage of antral follicles <2 mm at 4, 6, 8, and 10 weeks of age were significantly greater than the percentage of follicles ≥2 and <3 mm and the contribution of follicles ≥3 mm was the lowest. FSH levels increased slowly from birth to 6 weeks of age then, increased rapidly to week 10, followed by a decline at weeks 12 and 14. LH was low at birth and the level increased slowly to 8 weeks of age, followed by a further rapid increase at 10 weeks of age. All parameters studied did not show any significant differences between the right and left ovary. It was concluded that isofolliculia occurred between 4 and 10 weeks of age in left and right ovaries of Ouled Djellel ewe lambs. This phenomenon was characterized by the increase of both ovarian weights and dimensions, and of plasma FSH and LH levels. All large antral follicles ≥1 mm in diameter contributed to isofolliculia but the contribution of antral follicles <2 mm was greater than the contribution of antral follicles ≥2.     

Developmental pattern of small antral follicles in the bovine ovary

The study was designed to characterize the developmental pattern of 1- to 3-mm follicles and to determine the stage at which the future dominant follicle first attains a size advantage among its cohorts. In experiment 1, heifers (n = 18) were examined every 24 h by transrectal ultrasonography for one interovulatory interval (IOI). In experiment 2, cows (n = 9) were examined every 6 h from 5 to 13 days after ovulation to monitor precisely the diameter changes of individual follicles >/=1 mm during emergence of wave 2. Results revealed a change over days (P < 0.05) in the number of 1- to 3-mm follicles, with a maximum (P < 0.05) 1 or 2 days before wave emergence (conventionally defined as the time when the dominant follicle is first detected at 4 mm), followed 3-4 days later by a maximum (P < 0.05) in the number of >/=4-mm follicles. The profiles of small (1-3 mm) and large (>/=4-mm) follicles were inversely proportional (r = -0.79; P = 0.01). The profile of the number of 1- to 3-mm follicles during wave emergence was similar (P = 0.63) between waves in two-wave IOI, but differed (P < 0.01) among waves in three-wave IOI as a result of a greater number of follicles in the ovulatory wave (P < 0.04). As well, the number of follicles in the ovulatory wave tended to be greater (P < 0.06) in three-wave IOI than in two-wave IOI. The future dominant follicle was first identified at a diameter of 1 mm and emerged 6-12 h earlier than the first subordinate follicle (P < 0.01). After detection of the dominant follicle at 1 mm (0 h), its diameter differed from that of the first and second subordinate follicles at 24 h (P = 0.04) and 12 h (P = 0.01), when the dominant follicle was 2.4 +/- 0.17 mm and 1.7 +/- 0.14 mm, respectively. The growth rate of the dominant follicle differed from that of the first and second subordinate follicles at 120 h (P = 0.03) and 108 h (P = 0.02), when the dominant follicle was 9.5 +/- 0.30 mm and 8.8 +/- 0.49 mm, respectively. Emergence of the future dominant (r = 0.71), first (r = 0.73), and second (r = 0.76) subordinate follicles was temporally associated (P < 0.01) with a rise in circulating concentrations of FSH. Transient, nocturnal elevations in plasma FSH concentration were followed within 6 h by an increase in the growth rate of 1- to 3-mm follicles. We conclude that 1) 1- to 3-mm follicles develop in a wave-like manner in association with surges in plasma concentrations of FSH, 2) 1- to 3-mm follicles are exquisitely responsive to transient elevations in FSH, and 3) selection of the dominant follicle is manifest earlier than previously documented and is characterized by a hierarchical progression over a period encompassing the entire FSH surge (5 days).     

Role of intrafollicular regulators and FSH in growth and development of large antral follicles in sheep

Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11-12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal-early follicular phase of the cycle. Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12-15. Inhibition of folliculogenesis was represented by a decrease in those follicles greater than 4 mm, an increase in the relative proportion of follicles less than 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles less than 2 mm. There was no change in the extent of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of androstenedione on the growth and meiotic competence of bovine oocytes from early antral follicles

Summary Medium that contains 17β-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte-granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17β-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 μm (n = 191). Either steroid was necessary for maintainance of the organization of oocyte-granulosa cell complexes over the 14-day culture period. In the 17β-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22-24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17β-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17β-estradiol, can maintain the viability of bovine oocyte-granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose.     

Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles

Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5–1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P < 0.05) than that observed after 14 days of culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P < 0.05) when 12 or 14 days of IVG culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P < 0.05) when pre-IVM culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P < 0.05) after 12 days. Blastocyst production rate for oocytes obtained after 12 days of IVG culture (24.5%) was greater (P < 0.05) than for oocytes obtained after 14 days (9.9%). In conclusion, 12 days IVG followed by pre-IVM culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

Vascular endothelial growth factor production in growing pig antral follicles

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.     

In vitro culture of bovine preantral follicles

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

Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles

The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2-0.5 mm diameter; and (II) 0.4-0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus-oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus-oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7-10 days (Group II). After this, cumulus-oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus-oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus-oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2-3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus-oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7-10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus-oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence.     

A stereological study of medium antral follicles during the bovine estrous cycle

Stereological methods were applied to bovine cumulus-oocyte complexes (COCs) in order to characterize them quantitatively during the estrous cycle. COCs from medium (4-8mm) antral follicles with a compact and complete cumulus mass, and with an uniform or a non-visible ooplasm were aspirated from ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphological types (C1-C3) of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Quantitative data show that COCs appear heterogeneous for all studied parameters. From metestrus to proestrus the volumes of COCs and oocytes remained constant, the volumes of oocytes and oocyte nuclei were correlated, the thickness of the outer zona pellucida decreased, and the relative numerical frequency of follicular type C3 cells increased. Results suggest that COCs from distinct estrus stages are structurally different, with type C3 follicular cells gradually differentiating from cell types C1 and C2.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles.

This study investigated the capacity of healthy oocytes derived from follicles of different size to undergo normal fertilization and early embryonic development in vitro and full-term development in vivo. Ovaries were collected from a local abattoir and dissected and classified as follows: group A, greater than 4-8 mm (large); group B, greater than 2-4 mm (medium); and group C, greater than 1-2 mm (small). Oocytes were isolated by puncturing the follicular wall and pressing of the follicle. Only healthy-looking cumulus-oocyte complexes (COC) were used for in vitro maturation. Oocytes were fertilized in vitro by frozen/thawed semen from one bull. Approximately one-fourth of all oocytes was fixed and stained 15-20 h after fertilization, to determine penetration rates. The remaining eggs were transferred to culture medium and were cultivated for up to 9.5 days. Cleavage was observed 65 h and 7 days after fertilization. Expanded, hatching, and hatched blastocysts were fixed and stained after 9.5 days of culture. A total of 86 blastocysts derived from group A and B oocytes was nonsurgically transferred to synchronized recipients 7-8 days after onset of culture. A total of 6.624 follicles were dissected from 265 ovaries, and 1,485 oocytes were isolated from 1,671 group A follicles, 3,509 oocytes from 3,862 group B follicles, and 965 oocytes from 1,091 group C follicles. The fertilization rate, rate of normal fertilization, rate of polyspermy, and rate of other abnormal fertilization features were as follows: group A, 84.9%, 43.2%, 34.1%, 7.6%; group B, 83.6%, 44.8%, 31.1%, 7.8%; and group C, 61.7%, 13.1%, 33.7%, 19.1%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.