Medium Follicles Research Articles

Flow cytometric cell cycle analysis of two subpopulations of porcine granulosa cells.

Two types of granulosa cells from 120 individual follicles (forty follicles in particular stages of development) were analysed by DNA flow cytometry to determine the percentage of cells with degraded DNA (apoptosis) and the cell cycle analysis. The distribution of cells in the cell cycle (G1, S, G2/M) was studied to show relation to location within a follicle and follicular development. Isolated granulosa cells were scraped from the vesicle walls with rounded-tip ophthalmological tweezers and separated into weakly-associated (AGc) and tightly-bound (MGc) according to Ford (1991) and Duda (1997). Granulosa cells after fixation in 70% ethanol and staining with propidium iodide (PI) were analysed. At least 20,000 events were collected in each specimen. The S-phase fraction (SPF) and apoptosis were calculated using the ModFit LT programme for the cell cycle analysis (Verity Software House Inc., USA). In AGc a population with degraded DNA was found, containing less fluorescence than the G1/G0 peak as shown in the DNA histograms. The percentage of apoptotic AGc ranged from 39.29 in small, to 58.9 in medium and was significantly higher than in large follicles (26.13%; p < 0.05). The percentage of apoptotic MGc was significantly lower than in the AGc (p < 0.05) and was equal to 3.78 in small, 0.10 in medium and 0.08% in large follicles. There are no significant statistical differences between the mean percentage of SPF in MGc of small and medium follicles (4.94, 7.25%). However, SPF was significantly lower in large follicles (1.31%). The number of SPF in AGc decreased during follicular development (35.92, 26.98, and 19.62%). Our data indicate lack of apoptotic cell death in MGc which seem to be more differentiated, and lose their mitotic potential. In AGc however, which are undifferentiated and undergo numerous mitosis, apoptosis was observed.

Expression of Follicle-Stimulating Hormone Receptor Variants During the Estrous Cycle of the Ewe.

Several splice-variants of the follicle stimulating hormone receptor (FSHR) have been identified in sheep. These include a G protein-coupled (FSHR-1), a putative dominant negative (FSHR-2), and a growth factor type-I (FSHR-3) form. These variants likely couple to different signaling pathways and may be involved in FSH responsiveness at different stages of follicular development. However, with the exception of FSHR-1, the roles of variant forms of the FSHR during follicular development in the ewe are unknown. Thus, our objective was to examine expression patterns of variant forms of the FSHR at different times during the estrous cycle and in different sized follicles in the ewe. In Exp. 1, 8 cycling Suffolk-cross ewes were euthanized 24 (n=3), 36 (n=3) and 48 (n=2) hr after the onset of estrus. In Exp. 2, 7 anestrous Suffolk-cross ewes received CIDRs for 14d, followed by treatment (n=3) with PMSG (500IU), or no PMSG (n=4; CIDR-only). Ewes were euthanized 24 (n=4; 2 CIDR-only, 2 PMSG-treated) or 36 (n=3; 2 CIDR-only, 1 PMSG-treated) hr after CIDR removal. Ovaries were harvested from each ewe and all visible follicles were aspirated. Granulosa cells (GC) were isolated from follicular fluid; each isolate was pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm) or preovulatory (≥ 6.1 mm). Total RNA was extracted from GC and reverse-transcribed. The resulting cDNA was subjected to qPCR using primers designed to amplify each FSHR variant specifically. For Exp. 1, FSHR-3 was the most highly expressed (P≤0.05) variant in small and medium follicles. Expression of FSHR-2 and FSHR-3 was higher (P≤0.05) in medium follicles compared to small and preovulatory follicles. Expression of FSHR-1 was not significantly different between follicle size classes, but was higher (P≤0.05) than FSHR-2 in small follicles. No differences in FSHR variant expression were found at different times after onset of estrus, nor were FSHR variants expressed at significantly different levels in preovulatory follicles. For Exp. 2, application of CIDRs during anestrous appeared to cause slight differences in expression patterns of FSHR variants. The most highly expressed variant (P≤0.05) in small and medium follicles was still FSHR-3; however, FSHR-1 expression was higher (P≤0.05) in large versus small and medium follicles, while no differences were found in experiment 1 for FSHR-1 in different sized follicles. Thus, progesterone treatment during anestrous may enhance expression of the G protein-coupled form of the FSHR as follicles develop. FSHR-2 was expressed at the lowest (P≤0.05) levels of any variant, but was higher (P≤0.05) in large compared to medium and small follicles. No significant differences in FSHR variant expression were found within large follicles. This is the first study to examine expression patterns of FSHR-1, FSHR-2, and FSHR-3 during development of follicles in the mature sheep ovary. Our results indicate that FSHR-3 is the predominant variant of the FSHR expressed by GC during the ewe estrous cycle, and thus FSH may have important effects on follicular development through other forms of its receptor in addition to the G protein-coupled form. (poster)

Follicular Population during the Oestrous Cycle in Nili-Ravi Buffaloes Undergoing Spontaneous and PGF2a Induced Luteolysis

The objective of this study was to compare the follicular population during spontaneous and PGF 2α induced oestrous cycles in Nili-Ravi buffaloes. In Exp.1, (n = 13 oestrous cycles) follicular population was monitored using ultrasonography on alternate days. Buffaloes were monitored for ovarian follicles from day 0 (first oestrus) until next oestrus. These animals were observed for oestrus twice daily using a teaser bull. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves during the oestrous cycle. The mean number of small, medium and large follicles among various days of the oestrous cycle between two and three waves of follicular development were not significantly different (p>0.05). In Exp. 2, follicular population 3 days before oestrus was compared in buffaloes undergoing spontaneous (n = 12 oestrous cycles) and PGF 2α induced (n = 6) luteolysis. The mean number of small and large follicles increased (p<0.05) and the number of medium follicles decreased (p<0.05) during the 3 days before oestrus in buffaloes undergoing induced luteolysis as compared to those with spontaneous luteolysis. These results showed that the mean number of small, medium and large follicles among various days of the oestrous cycle were similar between the two and three waves of follicular development, and three days before oestrous the number of small, medium and large follicles altered due to induced luteolysis on day 9, compared to those with spontaneous luteolysis.

Differences in preovulatory follicle dynamics and timing of preovulatory LH surge affect fertility of maiden sheep reared in semi-arid extensive conditions

In the current study follicular dynamics, pituitary function, ovulatory response and luteal activity of 30 maiden Barbarine sheep were analyzed according to oestrus occurrence and lambing outcome after oestrus synchronisation with cloprostenol. Animals were retrospectively classified in three groups named as O− ( n = 7, ewes not displaying oestrus), O+L− ( n = 7, ewes showing oestrus but failing to lamb) and O+L+ ( n = 16; ewes showing oestrus and lambing thereafter). All the sheep ovulated and daily transrectal ultrasonographies revealed that preovulatory follicles were present at cloprostenol injection in all the animals. In sheep O+L+ and O+L−, 50% and 57% of the ovulatory follicles were the largest follicles at cloprostenol treatment (mean size of 4.1 ± 0.26 mm and 4.3 ± 0.74 mm, respectively). In O− ewes, the same percentage was higher (86%, P < 0.05 when compared to group O+L+; mean size of 4.0 ± 0.46 mm). The number of large follicles and the final diameter of the ovulatory follicles at oestrous tended thereafter to be higher in group O+L+ (1.4 ± 0.1 and 6.4 ± 0.2) than in groups O+L− (1 ± 0.2 and 5.7 ± 0.36) and O− (0.9 ± 0.2 and 5.9 ± 0.5, respectively). Conversely, the number of medium follicles at oestrus detection was higher in the group O+L− (2.1 ± 0.3, P < 0.05) than in the other two groups (1 ± 0.2 and 1 ± 0.3 for O+L+ and O− respectively). Timing of preovulatory LH surge was earlier for ewes O− (24.0 ± 4.75, P < 0.05) than for sheep O+L+ and O+L− (37.9 ± 2.45 h and 38.0 ± 4.75 h, respectively) and 94% of O+L+ ewes had a LH surge between 16 h and 64 h after cloprostenol injection compared to 57% in O+L− and O− groups ( P < 0.05). Thus, maiden Barbarine sheep failing to display oestrus or conceive showed alterations in their follicular dynamics and, thereafter, pituitary function and ovulatory response.

Follicular Size is Associated with the Levels of Transcripts and Proteins of Selected Molecules Responsible for the Fertilization Ability of Oocytes of Puberal Gilts

The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (BCB) test, real-time quantitative PCR reaction methods, western-blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts.

519. INFLUENCE OF THE DURATION OF CIDR INSERTION ON OVARIAN RESPONSES IN AN eCG PROGRAM DURING NON-BREEDING SEASON IN EWES

The aim of this study was to investigate the effect of 14 versus 7 days CIDR insertion before eCG treatment on superovulatory responses during non-breeding season in ewes. 10 Iranian Shall ewes, between 2 and 3 years old were kept inside during the experiment period and were fed a live weight maintenance ration. Ewes were randomly assigned to 1 of 2 groups. CIDR was inserted to the ewes of group 1 for 14 days and for 7 days to group 2. Each ewe received 1500 IU eCG on the day of CIDR removal. Ovarian follicular activity was monitored by transrectal ultrasonography on the days of eCG treatment, estrus, and 7 days after estrus to monitor the number of corpus lutium. During examination the animals were held in a standing position. Scanning of both ovaries was recorded using a MP4 player. The number of medium and large follicles (≥3 mm in diameter) were recognized, measured and mapped to their location using printed images of both ovaries. Data were analyzed using GLM procedure of the SAS. The results presented in Table 1 shows that the ovarian responses in terms of the number of ≥3 mm follicles at estrus is greater (P&lt;0.05) in group 1 than group 2 ewes. The mean number of CL after eCG injection in group 1 and 2 were also significantly different (3.6±0.24 vs 2.4±0.51; P&lt;0.05). The results showed that the 14 days synchronization before eCG treatment during non-breading seasons in ewes had the beneficial effects on ovarian responses.

516. THE EFFECTS OF eCG INJECTION ON SUPEROVULATORY RESPONSES BEFORE VERSUS AFTER CIDR REMOVAL IN IRANIAN SHALL EWES

The aim of this study was to investigate the effects of eCG injection on ovarian responses 2 days before or immediately after CIDR removal in a superovulation program during non-breeding season in Iranian Shall ewes. 12 ewes were kept inside for the duration of the experiment and were fed a live weight maintenance ration. The ewes were synchronized by CIDR. The day of CIDR insertion was considered as the starting point of the experiment (day 0). The ewes received 1500 IU eCG on day 12. Ewes were randomly assigned to 1 of 2 groups, and CIDR removed on days 12 and 14 in groups 1 and 2, respectively. Ovarian follicular activity was monitored by transrectal ultrasonography daily from the initiation day of superovulatory stimulation (day 12) until the day of estrus (day 14), and once 7 days after estrus (day 21) to monitor the number of corpus lutium. During examination the animals were held in a standing position. Ultrasonographic scanning of both ovaries was recorded using a MP4 player. The number of medium and large follicles (≥3 mm) were recognized, measured and mapped to their location using printed images of both ovaries. Data were analyzed using GLM procedure of the SAS. The results presented in Table 1 showed that the ovarian responses in terms of the number of ≥3 mm follicles at estrus and CL 7 days later were greater (P&lt;0.05) in group 1 ewes. The results indicated that the superior time for eCG injection is on the day of CIDR removal.

520. RELATIONSHIP BETWEEN THE OVARIAN STATUS ON THE INITIATION DAY OF SUPEROVULATION AND THE OVULATORY FOLLICLES AT ESTRUS IN SHALL IRANIAN EWES

The objective of this experiment was to determine the influence of the ovarian status on the initiation day of superovulation (day 14) on superovulatory response in terms of the number of ovulatory follicles (F, ≥4mm) at estrus (day 16). Estrous cycles of 50 Shall Iranian ewes were synchronized by CIDR. The day of CIDR insertion was considered as the starting point of the experiment (day 0). At the time of CIDR removal (day 14), 650 IU of eCG was administered to ewes. Ultrasonography was performed on days 14 and 16. Data were analyzed by means of the GLM procedure of the SAS. In these cases, the number of follicles were determined in each of the three studied classes (&lt;2, 2 to 4, and ≥4 mm) on day 14. Subsequently, each of these classes was divided into two subgroups (Low, High) based on the number of follicles observed on day 14 (Table 1). The number of large follicles at estrus in each of the three studied classes were (3.96±0.28 vs. 4.78±0.26, p&lt;0.05; 4.31±0.35 vs. 4.41±0.24, p&gt;0.05; and 4.50±0.23 vs. 4.07±0.37, p&gt;0.05) respectively. The number of medium and large follicles on day 14 did not affect the superovulatory response. However, the high number of small follicles on the initiation day of superovulation increased the number of ovulatory follicles at estrus. Since, 23 out of 50 ewes had a low number of small follicles on day 14 (Table 1), which may suggest that an injection of FSH 1-2 days before initiation of eCG treatment enhance the superovulatory response.

296 EFFECT OF GnRH AND ESTRADIOL BENZOATE INJECTION ON SUPEROVULATORY RESPONSE IN EARLY POSTPARTUM SUCKLING JAPANESE BLACK COWS

The objective of this study was to investigate the effect of GnRH and EB injection on the SOV response in early postpartum suckling Japanese Black cows. Forty-eight cows were included in this study. All cows received a controlled internal-drug-releasing device containing 1.9 g progesterone (CIDR; Easi-Breed; InterAg, Hamilton, New Zealand) at 40 days postpartum, and were subsequently superstimulated with a total dose of 20 armour units FSH (Antrin 40; Kawasaki-Mitaka, Kanagawa, Japan) twice daily, with gradually decreasing doses from day 45 till day 47. CIDR was withdrawn with 0.75 mg of cloprostenol (Estrumate; Nagase Medicals, Itami, Japan) injection at day 47. After FSH treatment, the cows were assigned to 3 groups: (1) 100 μg GnRH (Conceral; Nagase Medicals) injection at day 49 (GnRH, n = 19), (2) 2 mg EB (Kawasaki-Mitaka) injection at day 48 (EB, n = 15), and (3) control (n = 14). AI was carried out twice based on the estrous behavior of the cows, which was examined several times daily. Embryos were nonsurgically collected 7 to 8 days after estrus. The ovaries were examined by ultrasonography, and the number of CL and unovulated follicles (UF) were counted. After uterine flushing, the cows were re-employed for reproductive purposes. The intervals to first estrus after flushing and days open were examined. Blood samples were collected temporally, and concentrations of progesterone (P4) and estradiol (E2) were measured. The ovaries were examined by ultrasonography and the number of CL, small follicles (&lt;5 mm in diameter), medium follicles (between 5 and 10 mm in diameter), and large follicles (&gt;10 mm in diameter) were counted temporally. Data were analyzed by GLM and Dunnett’s T3 test. There were no significant differences between both treatment groups and the control group in the number of CL (15.2 ± 6.8, 18.9 ± 9.4, and 14.9 ± 8.9 in the GnRH, EB, and control groups, respectively; mean ± SD), UF (5.3 ± 3.2, 4.7 ± 3.2, and 7.2 ± 9.5, respectively), recovered ova or embryos (11.4 ± 8.1, 11.1 ± 8.9, and 11.1 ± 9.1, respectively), transferable embryos (6.8 ± 5.3, 7.5 ± 7.6, 7.2 ± 6.7, respectively), and freezable embryos (6.3 ± 5.6, 6.6 ± 7.9, and 5.7 ± 6.1, respectively). Furthermore, there were no significant differences in the intervals to first estrus after flushing and days open (12.7 ± 13.6 and 90.0 ± 23.1 in the GnRH, 8.6 ± 5.3 and 79.1 ± 20.1 in the EB, and 9.4 ± 6.0 and 88.5 ± 33.8 in the control groups, respectively). Changes in the P4 level were similar among the 3 groups. The E2 level of the EB group at 15 h after EB injection was significantly higher (P &lt; 0.05) than that in the other groups (19.0 ± 15.7, 49.4 ± 22.8, and 8.0 ± 5.7 pg mL–1 in the GnRH, EB, and control groups, respectively). The number of small follicles in the control group (17.0 ± 12.2) was significantly greater (P &lt; 0.05) than that in the GnRH group (7.4 ± 7.2) at the first AI. These results suggest that EB and GnRH injection after SOV treatment in early postpartum Japanese Black cows does not affect the SOV response or embryo quality.

171 ADMINISTRATION OF GnRH ON DAY 6 ALTERS THE NUMBER OF FOLLICULAR WAVES FROM 2 TO 3 DURING THE ESTROUS CYCLE OF HOLSTEIN COWS

It is suggested that pregnancy rate is greater in lactating cows inseminated following ovulation of a third-wave follicle compared with a second-wave follicle. The number of follicular waves is not apparent during the estrous cycle. However, GnRH injection on Day 6 is supposed to initiate a new follicular wave earlier; as a result, the number of cows with 3 follicular waves will be increased. This study was done to change the 2-follicular-wave cycles to 3 follicular waves during the estrous cycle. The estrous cycles of 10 cows were synchronized with 2 i.m. injections of prostaglandin F2α given 11 days apart. The cows were randomly assigned to 1 of 2 treatments. Cows in the control treatment received no treatment, whereas GnRH6 cows received a GnRH injection on Day 6 of the estrous cycle (estrus = Day 0). Ovaries of all cows were scanned daily, from estrus (Day 0) to the subsequent estrus (Day 23). Profiles of the mean number of follicles (i.e. 4- to 6-mm and ≥7 mm), the diameter of the largest follicle (F1), and comparison of 2- and 3-wave cows were compared by least squares analysis of variance using the general linear model (GLM) procedure of SAS. The number of 4 to 6 mm follicles had a tendency to decline until Day 6 (3.0 ± 2.1 v. 0.8 ± 1.6). In response to an injection of GnRH, the number of small follicles in group GnRH6 on Day 6 (1.5 ± 1.6 follicles) increased (P &lt; 0.05) on Day 8 (5.0 ± 1.7 follicles) and the number of medium and large follicles (≥7 mm) on Day 6 (1.8 ± 0.5 follicles) decreased (P &lt; 0.05) on Day 9 (0.8 ± 0.6 follicles). Over the 4-day period before GnRH treatment, the diameter of F1 was increasing (from 4.7 ± 0.9 mm to 10.0 ± 0.6 mm) in cows treated with GnRH on Day 6 of the estrous cycle; thereafter, the diameter of the F1 decreased (P &lt; 0.05) within 2 days of GnRH treatment. In the control group the diameter of the F1 increased over the 4-day period before GnRH treatment (from 5.0 ± 0.9 mm to 11.2 ± 0.6 mm), whereas it remained constant (11.2 ± 1.2 mm to 11.2 ± 1.2 mm) between Days 6 and 9 of the estrous cycle (P &lt; 0.003). Results showed that 4 out of 5 cows in control group had 2 follicular waves, whereas all of the cows (n = 5) in GnRH6 group had 3 follicular waves. The first follicular wave begins at Day 1.1 ± 0.50 and 0.70 ± 0.50 in the control and GnRH groups, respectively. The second follicular waves appeared at Day 11.00 ± 0.80 and 8.30 ± 1.50 (P ≤ 0.05) for the 2- and 3-wave animals, respectively. The third follicular waves (n = 6) initiated at Day 16.30 ± 1.50. Cows with 2 follicular waves had shorter estrous cycles (P ≤ 0.05) than 3-wave cows (20.50 ± 0.60 v. 22.40 ± 0.90). The ovulatory follicle in 2-wave cows grew larger (14.00 ± 1.80 v. 12.50 ± 0.90 mm; P ≤ 0.05), and maintained for a longer period of time (9.50 ± 1.80 v. 6.1 ± 0.90; P ≤ 0.05) than in 3-wave cows. In conclusion, an injection of GnRH on Day 6 of the estrous cycle could promote the emergence of a new follicular wave earlier in cows and alter the estrous cycles with 2 waves into 3 follicular waves.

Physiological mechanisms of ovarian follicular growth in pigs — A review

Follicular growth after antrum formation is determined by follicle-stimulating hormone (FSH). Only two ways are possible for recruited follicles, continuing development or atresia. In gilts, intensive ovarian follicular growth begins between 60 and 100 days of age, and fluctuations of the ovarian morphological status last about 20 days; however, at that time there are no really large follicles. Final follicular development is under luteinising hormone (LH) control; this is why the attainment of puberty is related to an increase in serum oestradiol to a level that causes a preovulatory surge of this gonadotropin. The pool of follicles at the beginning of the oestrous cycle is about 30-40, most of which are small (< 3 mm) and growing. Then, the pool of follicles increases to about 80 in the mid-luteal phase but about 50 of them are small and 30 are medium sized (3-6.9 mm). Some of these follicles are in the growing phase, but some are atretic. Between days 7 and 15 of the oestrous cycle the percentage of atretic follicles fluctuates between 12 and 73%. At that time there are no large (> 7 mm) follicles because of the suppressing effect of progesterone. The number of small follicles declines after luteolysis. From the pool of medium follicles, large follicles are selected under the influence of LH, but about 70% of the medium-sized follicles become atretic. Because of the long-lasting selection process there is a significant heterogeneity in the diameter of large follicles in oestrus. However, the number of follicles correlates with the number of corpora lutea after ovulation. Individual follicular development and the relationship between follicles are still poorly known. The use of ultrasonography may give a closer insight into these phenomena.

Keratinization of outer root sheath cells is prevented by contact with inner root sheath of rat hair follicles

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered--inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca2+ content formed monolayers, which after elevation of Ca2+ produced multilayers with cells containing keratohyalin-like granules. Ca2+ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca2+ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.

Effect of overexpression of inhibin α (1–32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.

Effect of exogenous insulin administration on ovarian function, embryo/fetal development during pregnancy in goats

The effect of insulin was investigated on ovarian follicle population, ovulation rate, hormonal profiles and embryo/fetal development during pregnancy using transrectal ultrasonography in goats. Twelve goats synchronized in estrus were selected for the experiment. They were divided into two groups, viz. (untreated control, n = 6) and (insulin treated, n = 6). In treated group long acting bovine insulin was administered @ 0.2 IU/kg body weight subcutaneously for three consecutive days, i.e. days 7–9 of estrous cycle. Thereafter, weekly single injection of insulin was continued for rest of the experiment. However, in control group only normal saline was injected as placebo. Breeding was allowed by natural service in both the groups. The does were subjected to B-mode transrectal ultrasound scanning of ovary and uterus weekly up to 120 and 98 days of gestation, respectively. Blood samples were collected weekly up to 135 days of gestation for the estimation of estradiol 17β and progesterone (P4). The result revealed no difference in mean number of total follicles between the control and insulin treated groups. The diameter of medium follicle did not differ where as diameter of large follicle was comparatively higher in treated than control goats. The average number of corpus luteum (CL) was higher in insulin treated group as compared to control (1.66 vs. 1.16). However, the number as well as mean diameter of CL did not differ significantly between treated and control group. Serum concentrations of estradiol 17β and progesterone were significantly ( P < 0.01) higher in treated than control goats. Embryonic vesicle was detected by day 21 in both the groups, however, its diameter did not differ significantly (0.73 and 0.72 cm) between the groups. The twinning percentage was higher (50 vs. 16%) in insulin treated than the control goats. Placentome diameter was also higher ( P > 0.05) in treated animals. The results demonstrated beneficial effect of exogenous administration of insulin on ovarian function and twinning percentage in goats.

A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin

The objective of this study was to determine whether follicles grown within human ovarian cortical strip culture for 6 days in serum-free medium could be isolated at the secondary stage of pre-antral development and grown in vitro to the late pre-antral/early antral stage during a 4 day culture period. Ovarian cortical biopsies were obtained from six women aged 26-40 years, with informed consent, during elective Caesarean section. Small tissue slices of ovarian cortex, with underlying stromal tissue removed, were cultured in serum-free medium for 6 days and at the end of this period pre-antral (secondary) follicles were dissected from the strips. Seventy-four intact pre-antral follicles ranging in size (66-132 microm) (mean size 100 microm +/- 3.4) were selected for further culture. Follicles were placed individually within V-shaped microwell culture plates in serum-free medium in the presence (n = 38) or absence (n = 36) of 100 ng/ml of human recombinant activin A. Pre-antral follicles grown for 4 days in the presence of activin A grew to a larger size (mean diameter 143 microm +/- 7.4) than those grown in control medium (mean diameter 111 microm +/- 8) (P < 0.005). Ninety percent of follicles cultured in the presence of activin A increased in size during the first 2 days of culture compared with only 36% of follicles in control medium (P > 0.005). Of the follicles surviving the entire culture period, 30% of those cultured in the presence of activin A showed normal morphology with intact oocytes and antral formation. None of the follicles grown in control medium developed antral cavities and >90% of those follicles collected at the end of the culture period showed signs of oocyte degeneration. The results reported here demonstrate that under certain conditions, it is possible to achieve accelerated oocyte/follicle development from human primordial/primary follicles. This provides the first encouraging step towards achieving full in vitro growth of human oocytes.

Oestrous cycle stage influences the morphology and maturation of porcine oocytes in vitro

The objective of the study was to characterize the effect of the oestrous cycle stage on the yield, morphology and meiotic competence of porcine oocytes. A total of 46 cycling gilts, at 8.5–9 months of age, were used as oocyte donors. Their oestrous cycle was synchronized by Regumate and the onset of oestrus was checked (Day 0). The gilts were slaughtered at the early (Days 1–5), middle (6–10) and late (11–14) luteal or early (Days 15–16), middle (17–19) and late (20–21) follicular phase. Oocytes were isolated separately from medium (5–9 mm) and small (≤ 4 mm) follicles. Cumulus-oocyte complexes with dark, evenly granulated cytoplasm and at least two compact layers of cumulus cells were selected as useful for maturation. They were matured by a standard protocol, denuded from cumulus cells, fixed in glutaraldehyde, stained with 33258-Hoechst and examined by epifluorescence. The oocytes collected from small and medium follicles differed in their yield, morphology and meiotic competence regardless of the phase. The mean number (± S.E.M.) of oocytes isolated per donor was higher (187.7 ± 48.4 vs. 16.9 ± 6.1) but the mean percentage (± S.E.M.) of useful oocytes was lower (22.4 ± 7.5% vs. 80.2 ± 6.8%; &lt;I&gt;P&lt;/I&gt; &lt; 0.01) for small than for medium follicles. The mean number (± S.E.M.) of useful oocytes per donor was significantly (&lt;I&gt;P&lt;/I&gt; &lt; 0.01) higher (42.1 ± 16.8 vs. 11.9 ± 4.3) but the mean percentage of matured oocytes was significantly (&lt;I&gt;P&lt;/I&gt; &lt; 0.01) lower (48.4 ± 17.8% vs. 79.9 ± 7.9%) for small than for medium follicles. The oocyte population collected from small follicles varied during the oestrous cycle. The mean number (± S.E.M.) of oocytes isolated per donor from small follicles increased during the luteal and decreased during the follicular phase, except for the late follicular phase when it increased again. The mean percentage (± S.E.M.) of useful oocytes did not differ too much during this period, except for the late follicular phase when it decreased significantly (&lt;I&gt;P&lt;/I&gt; &lt; 0.01). The mean number (± S.E.M.) of useful oocytes per donor increased during the luteal and decreased during the follicular phase, but the differences were not significant except for the late follicular phase (&lt;I&gt;P&lt;/I&gt; &lt; 0.01). Similarly, the mean percentage (± S.E.M.) of matured oocytes increased during the luteal and decreased during the follicular phase, and the differences were significant. Compared with the oocyte population from small follicles, the oocyte population from medium follicles was less variable in the period from the middle luteal to middle follicular phase, when these follicles were present on the ovaries. It can be concluded that the porcine oocyte population changes in terms of quantity, morphological quality and meiotic competence according to the stage of follicular development. The late luteal and early follicular phases appeared to be most productive for oocyte recovery, because more morphologically normal oocytes with greater meiotic competence were collected, as compared with the other stages of the oestrous cycle.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 α-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion‐enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.

Intrafollicular Concentrations of Steroid Hormones and PGF2α in Relation to Follicular Development in the Mares during the Breeding Season.

The concentrations of androstenedione, estradiol-17β, progesterone and PGF2α contained in the follicular fluid produced by the follicles in collected ovaries of mares that have had estrous phase during the breeding season were measured and analyzed the relation between the growth stage of follicles and the hormone levels in the follicular fluid. An ultrasonographic diagnostic instrument was used to measure the diameter of the follicles in order to categorize the follicles into three groups the following: 8 small follicles (from 1.0 to less than1.5 cm), 8 medium follicles (from 1.5 to less than 3.0 cm), and 8 large follicles (from 3.0 to 5.0 cm), respectively. The analysis of the follicular fluid in ovaries of estrous mares showed that the concentrations of androstenedione were significantly higher in the medium or large follicles than in the small follicles and the concentrations of estradiol-17β were significantly higher in larger follicles than in the small or medium follicles (P<0.05). The concentrations of progesterone and PGF2α, on the other hand, did not significantly vary regardless of follicluar size. In the follicles within the mare ovaries that have had estrous stage, the concentrations of the hormones related the ovulation, namely androstenedione and estradiol-17β, were higher with larger follicles.

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.