Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.     

Pattern of growth of dominant follicles during the oestrous cycle of heifers

Ovarian follicular development was studied in 13 heifers by daily ultrasound examination during 2 complete and consecutive natural oestrous cycles. In 21 cycles (81%) 3 dominant follicles were identified, in 4 cycles (15%) 2 and in the remaining cycle 1 (4%). Consistently, the first dominant follicle was detected on average on Day 4, reached a maximum size on Day 6, went through a period of relative stability between Days 6 and 10, then began to decrease in size and was undetectable by Day 15. The second dominant follicle was detected by Day 12, reached maximum size on Day 16 (or 19 in the 4 cycles in which the 2nd dominant follicle was the ovulatory follicle) and was undetectable by Day 19. The 3rd (ovulatory) follicle was identified on average by Day 16 (range Days 10 to 19) and maximum size was reached on Day 21. The ovulatory follicles were larger (P less than 0.05) than the previous ones and the stage of the cycle at which maximum size was reached was significantly different for each dominant follicle (P less than 0.05). The analysis of the rates of growth and atresia suggest that the rate of growth is slowest during mid-cycle. The number of dominant follicles that developed in the ovary ipsilateral to the corpus luteum was greater (P less than 0.05) than in the contralateral ovary.     

Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers

Previous studies demonstrated that waves of follicular activity develop approximately every 9 d in cattle during the estrous cycle and early pregnancy. A dominant follicle develops from each wave and the remaining follicles (subordinates) begin to regress after a few days. In this study, intraovarian luteal and follicular interrelationships were examined during the follicular waves of the estrous cycle and pregnancy using data obtained by ultrasonography. During the estrous cycle, no intraovarian relationships were found between the ovary containing the corpus luteum and the ovary containing the dominant follicle (n = 165), or between the location of the corpus luteum and the characteristics of the dominant follicle. During pregnancy, however, the frequency distribution for the number of follicular waves with the dominant follicle and corpus luteum on the same or opposite ovaries differed (P<0.05) among Waves 1 to 10. The two structures (dominant follicle and corpus luteum) were more often in opposite ovaries during Waves 3 to 10 (combined frequency, 75%) than during Waves 1 and 2. During pregnancy, dominant follicles of consecutive waves differed (P<0.05) among Waves 1 to 8 in the frequency with which they appeared in the same versus the opposite ovary. The difference seemed primarily due to an increased frequency of consecutive follicles on the same ovary for Waves 4 to 8 (combined frequency, 80%). During both the estrous cycle and pregnancy, there was no significant intraovarian effect of the dominant follicle on the day of detection of the next dominant follicle, on the growth rate of the largest subordinate follicle, or on the length of the interval from wave origin to cessation of growth of the largest subordinate; these results indicate that previously postulated suppressive effects between follicles are exerted through systemic channels.     

Suppression of dominant and subordinate ovarian follicles by a proteinaceous fraction of follicular fluid in heifers

Nulliparous Holstein heifers were examined ultrasonically once daily during an interovulatory interval (ovulation = Day 0). Follicles with a diameter >/=4 mm were sequentially identified. Heifers were randomized into four groups (n = 4 heifers per group): untreated control heifers and those treated on Days 0 to 3, Days 3 to 6, or Days 6 to 11. Heifers designated for treatment were given an intravenous injection, twice daily, of a proteinaceous fraction of follicular fluid (PFFF; 16 ml) prepared by extracting bovine follicular fluid with activated charcoal. Mean cessation of growth of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 5.5) than in heifers treated on Days 0 to 3 (Day 1.5) or Days 3 to 6 (Day 3.5). Mean onset of regression of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 12.0) than in heifers treated on Days 0 to 3 (Day 5.0) or Days 3 to 6 (Day 7.5). Mean cessation of growth of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 3.0) than in heifers treated on Days 0 to 3 (Day 1.2). Mean onset of regression of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 7.0) than in heifers treated on Days 0 to 3 (Day 4.8). In heifers treated on Days 6 to 11, cessation of growth and onset of regression of the dominant follicle (means, Days 5.2 and 12.0, respectively) were not significantly different from those of the controls. The hypothesis that PFFF treatment on Days 0 to 3 would cause suppression of all follicles of Wave 1 was supported. The hypothesis that PFFF treatment on Days 3 to 6 would not alter growth of the dominant follicle of Wave 1 was not supported. The mean day of detection of the dominant follicle of Wave 2 was different (P<0.005) in control heifers (Day 8.5) than in heifers treated on Day 0 to 3 (Day 5.5) or Days 6 to 11 (Day 14.2). The mean length of the interovulatory interval was shorter (P<0.05) in control heifers (20.5 d) than in heifers treated on Days 6 to 11 (23.2 d). The hypothesis that PFFF treatment on Days 6 to 11 would delay the emergence of Wave 2 was supported. The proportion of heifers with 2-wave interovulatory intervals was 3 4 for control heifers and 0 4 , 1 4 , and 4 4 for heifers treated on Days 0 to 3, Days 3 to 6, and Days 6 to 11, respectively (3 4 vs 0 4 , P<0.05); the remaining heifers had 3-wave interovulatory intervals. On average, in PFFF-treated heifers, follicles stopped growing 1 d after treatment was started, and Wave 2 was detected 3 d after treatment was stopped.     

Oocyte size and intrafollicular position in polyovular follicles in rabbits

Healthy follicles with 2-24 oocytes were observed in adult rabbit ovaries during all phases of folliculogenesis from primary to preovulatory follicles. Most follicles contained 2-3 oocytes which developed according to their topographical situation in the follicle. The central oocyte in a normal topographical situation has an almost normal growth and development up to metaphase II and cumulus expansion. The peripheral oocytes grow more slowly: most do not attain the normal size or resume meiosis and remain surrounded by ordinary granulosa cells. When the number of oocytes is higher than 3, the peripheral oocytes develop even more slowly, as do the central ones. It demonstrates the necessity for the oocyte to occupy a certain position inside the follicle and to reach a size which allows resumption of meiosis; the cumulus responds only to oocytes of normal size and position. We suggest that, despite the relative frequency of binovular follicles, fertilization of two oocytes originating from one follicle is unlikely.     

Effect of dietary intake on pattern of growth of dominant follicles during the oestrous cycle in beef heifers

Friesian x Hereford heifers (n = 19; mean +/- s.e.m. body weight (BW) = 375 +/- 5 kg) were used in a randomized incomplete block design. Heifers were fed 0.7 (n = 7; L), 1.1 (n = 7; M) or 1.8% (n = 5; G) of BW in dry matter (DM)/day for 10 weeks. Ovaries were examined by ultrasound, for one oestrous cycle, from week 5 of treatment. Maximum diameter of dominant follicles was smaller (P less than 0.05) in L (11.8 +/- 0.1 mm) than in M (13.7 +/- 0.2 mm) or G (13.2 +/- 0.3 mm) heifers. Growth rate (mm/day) of dominant follicles during the oestrous cycle was not affected (P greater than 0.05) by dietary intake. Persistence of dominant follicles was shorter (P less than 0.05) in L (9.8 +/- 0.2 days) than in M (11.9 +/- 0.3 days) or G (12.7 +/- 0.4 days) heifers. Three dominant follicles were identified during the oestrous cycle of 5 of 7 L, 3 of 7 M and 1 of 5 G heifers (P less than 0.10); 2 dominant follicles were identified in the remaining heifers (n = 2 of 7, 4 of 7 and 4 of 5, respectively). Length of the luteal phase and luteal-phase concentrations of progesterone were not affected (P greater than 0.05) by treatment. Low dietary intake reduced the diameter and persistence of dominant follicles during the oestrous cycle of beef heifers and tended to increase the proportion of oestrous cycles with 3 dominant follicles.     

Intraovarian effect of dominant follicle and corpus luteum on number of follicles during a follicular wave in heifers

The intraovarian relationships among dominant follicle (DF), corpus luteum (CL), and number of follicles between Days 0 to 5 (Day 0 = ovulation) in wave 1 (n = 65 waves) and Days 9 to 13 in wave 2 (n = 62) were analyzed in separate experiments in Bos taurus heifers. Ovaries were grouped into intraovarian patterns of DF–CL, DF alone, CL alone, and neither DF nor CL. In wave 1, the pattern frequencies of DF–CL or neither DF nor CL (34% each) were greater (P < 0.0004) than for DF alone or CL alone (16% each). The number of growing follicles ≥5.0 mm, was greater (P < 0.0001) in ovaries with the DF, even when the DF was removed from the tally (P < 0.03). In a factorial analysis of wave 1, there was a positive main effect of DF (3.9 ± 0.2 vs. 2.2 ± 0.2 follicles; P < 0.0001), but the main effect of CL and the interaction of DF and CL were not significant. In a factorial analysis of wave 2, there were more (P < 0.0001) follicles greater than 6 mm in ovaries with a DF when the DF was included and an approaching difference (P < 0.09) when the DF was excluded. The main effect of CL and the interaction of DF and CL were not significant. The hypothesis that both the DF and CL have a positive intraovarian effect on number of follicles in waves 1 and 2 was only partly supported; the DF, but not the CL, had an effect in the factorial analyses. Previous reports in cattle and sheep of a positive intraovarian effect of CL on number of follicles are questionable in that location of the DF was not considered.     

Follicular competition in cows: the selection of dominant follicles as a synergistic effect

Journal of Mathematical Biology - The reproductive cycle of mono-ovulatory species such as cows or humans is known to show two or more waves of follicular growth and decline between two successive...     

Stimulation of the largest subordinate follicle by intrafollicular treatment with insulin-like growth factor 1 is associated with inhibition of the dominant follicle in heifers

The effect of intrafollicular treatment of the second-largest follicle (F2) with insulin-like growth factor (IGF) 1 on the largest follicle (F1) and F2 was studied in heifers. Treatment of F2 was done when F1 reached ≥8.2 mm (expected beginning of follicle deviation; Day 0 or Hour 0). In each of two experiments, three groups (n = 6 or 7 heifers/group) were used: controls, F2 treated with vehicle and F2 treated with IGF1. The IGF1 treatment consisted of 200 μg of recombinant human IGF1 (pharmacological dose) in 20 μL of vehicle. In Experiment 1, the hypothesis that treatment of F2 with IGF1 has a stimulatory effect on F2 was supported by a greater (P < 0.05) incidence of F2 dominance (≥10 mm) in the IGF1 group (71%) than in the other two groups (8%), and a greater (P < 0.02) growth rate of F2 on Days 0-2. Unexpectedly, treatment of F2 with IGF1 had an inhibitory effect on F1, as indicated by a reduced (P < 0.03) growth rate of F1 during Days 0-1 and Days 0-4 and a lesser (P < 0.05) maximum diameter of F1 in the IGF1 group. In Experiment 2, the hypothesis of an inhibitory effect on F1 when F2 was treated with IGF1 was supported by a lesser (P < 0.04) increase in diameter of F1 and a lesser (P < 0.04) percentage of follicle wall with power-Doppler signals of blood flow between Hours 0 and 14 in the IGF1 group. Circulating concentrations of FSH and LH were not altered significantly in either experiment. In conclusion, treatment of F2 with IGF1 at the expected beginning of deviation had a stimulatory effect on F2, but an inhibitory effect on F1.     

Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles: a control mechanism for selection of dominant follicles

This review summarizes evidence for the role of proteolytic enzymes that degrade and inactivate insulin-like growth factor binding proteins (IGFBP) during follicular development in mammals. In some species (e.g., bovine), evidence indicates that decreases in IGFBP-4 and -5 levels in estrogen-dominant preovulatory follicles are likely due, in part, to increased protease activity, whereas lower levels of IGFBP-2 are not due to increased proteolysis. Increased IGFBP-4 and -5 protease along with lower amounts of IGFBP-4 binding activity and greater amounts of free IGF-I are some of the earliest developmental changes documented in bovine growing antral follicles. This protease activity has recently been ascribed to serine metalloprotease(s), including pregnancy-associated plasma protein-A (PAPP-A), which was first detected in human follicular fluid nearly 20 yr ago. Other recent studies verified the presence of PAPP-A mRNA in granulosa cells of humans, monkeys, cattle, mice, and pigs. Increases in the amount of PAPP-A mRNA in granulosa cells during follicular development occurs in some but not all species, indicating that other proteases or protease inhibitors may be involved in IGFBP degradation. Whether the hormonal control of PAPP-A production/activity by the ovary differs between monotocous and polytocous animals will require further study. These protease-induced decreases in IGFBP-4 and -5 likely cause increased levels of bioavailable (or free) IGFs that stimulate steroidogenesis and mitogenesis in developing dominant follicles, which ultimately prepare the follicle(s) and oocyte(s) for successful ovulation and fertilization.     

Acute dietary restriction in heifers alters expression of genes regulating exposure and response to gonadotrophins and IGF in dominant follicles

Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F(2α) (PGF) administration from heifers (n=25) that were individually fed a diet supplying 1.2 maintenance (M; control, n=8) or 0.4 M (restricted, n=17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n=11) or anovulatory (n=6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P<0.001) and insulin (P<0.05) in circulation; oestradiol (P<0.01) and IGF-I (P<0.01) in follicular fluid; and mRNA for FSHR (P<0.01) in granulosa cells but increased mRNA for IGFBP2 (P<0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P<0.05), IGF2 (P<0.01) and IGF1R (P<0.05) in granulosa cells and LHCGR (P<0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles.     

Oocyte morphology in dominant and subordinate follicles

The structure of oocytes aspirated from the dominant and its subordinate follicles was investigated from the achievement of follicular dominance to ovulation. Ovulation was induced in 18 heifers and 5 cows by injection of cloprostenol at days 8–14 (day 0 = day of ovulation), and follicular development was monitored by ultrasonography. The animals were slaughtered at days 3–11, but animals slaughtered on days 8–11 were given a second injection of cloprostenol at day 7 to allow ovulation of the dominant follicle of the first follicular wave. Oocytes were aspirated from the dominant (largest) and two largest subordinat efollicles and processed for transmission electron microscopy, whereas the follicular fluids were analyzed for concentrations of estradiol-17β (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. From days 3–7, the dominant oocytes had pronounced junctional contacts with the cumulus cells and a nonundulating nuclear envelope but showed an increase in the number of lipid droplets and a decrease in the size of Golgi complexes, the size of cortical granule clusters, and the number of microvilli stacks. After cloprostenol injection on day 7, but before the anticipated LH surge, the dominant oocytes showed a reduced oocyte cumulus contact, vacuolization of the nucleolus, undulation of the nuclear envelope, and dispersal of the mitochondrial clusters. The morphological alterations occurring in the dominant oocytes before the anticipated LH surge are suggested to be a prerequisite for the oocyte to achieve the competence to undergo final maturation. Subordinate follicles ceased growing at about days 3–4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The structural changes associated with oocyte degeneration showed similarities with the processes seen before and during final maturation of the dominant oocytes. © 1994 Wiley-Liss, Inc.     

Modulation of intrafollicular oestradiol in explanted hamster follicles does not affect oocyte meiotic status

Mature antral follicles were collected from PMSG-primed hamsters before the LH surge on Day 4 of the oestrous cycle and were incubated individually for 6 or 10 h under 95% O2 and 5% CO2. The relationship between follicle size and follicular fluid volume was established, and both the oestradiol output during incubation and the LH sensitivity of the follicle with respect to induction of meiotic resumption were determined. The data obtained from these initial studies were then applied to experiments in which the oestrogenic status of follicles was altered by pressure injection of solutions into the antral cavity to result in known concentrations in follicular fluid of oestradiol (0-500 microM), the anti-oestrogens, tamoxifen (0-200 microM) or enclomiphene citrate (0-500 microM), or an oestradiol-specific antiserum (1:200 dilution of serum). The proportion of immature oocytes (% GV) was determined cytogenetically after follicular incubation. Significant correlations were established between follicle size and both follicular fluid volume (r = 0.90) and follicular oestradiol accumulation (r = 0.96). The minimum concentration of LH tested which induced maximal meiotic resumption (0% GV) was 0.5 microgram/ml. The % GV was not significantly reduced by intrafollicular injection of either of the anti-oestrogens over the concentration ranges tested, or of the oestradiol-specific antiserum, which was injected in an amount adequate to bind all oestradiol accumulated during the incubation. Intrafollicular injection of oestradiol failed to increase the % GV after follicular incubation in the presence of 0.5 microgram LH/ml. From these results we conclude that it is unlikely that oestradiol plays a primary role in the maintenance of meiotic arrest in follicle-enclosed hamster oocytes in vitro.     

Differentiation of dominant versus subordinate follicles in cattle

Selection of a dominant follicle, capable of ovulating, from among a cohort of similarly sized follicles is a critical transition in follicular development. The mechanisms that regulate the selection of a species-specific number of dominant follicles for ovulation are not well understood. Cattle provide a very useful animal model for studies on follicular selection and dominance. During the bovine estrous cycle, two or three sequential waves of follicular development occur, each producing a dominant follicle capable of ovulating if luteal regression occurs. Follicles are large enough to allow analysis of multiple endpoints within a single follicle, and follicular development and regression can be followed via ultrasonographic imaging. Characteristics of recruited and selected follicles, obtained at various times during the first follicular wave, have been determined in some studies, whereas dominant and subordinate follicles have been compared around the time of selection in others. As follicular recruitment proceeds, mRNA for P450 aromatase increases. By the time of morphological selection, the dominant follicle has much higher concentrations of estradiol in follicular fluid, and its granulosa cells produce more estradiol in vitro than cells from subordinate follicles. Shortly after selection, dominant follicles have higher levels of mRNAs for gonadotropin receptors and steroidogenic enzymes. It has been hypothesized that granulosa cells of the selected follicle acquire LH receptors (LHr) to allow them to increase aromatization in response to LH, as well as FSH. However, LH does not appear to stimulate estradiol production by bovine granulosa cells, and the role of LHr acquisition remains to be determined. Recent evidence suggests a key role for changes in the intrafollicular insulin-like growth factor (IGF) system in selection of the dominant follicle. When follicular fluid was sampled in vivo before morphological selection, the lowest concentration of IGF binding protein-4 (IGFBP-4) was more predictive of future dominance than size or estradiol concentration. Consistent with this finding, dominant follicles acquire an FSH-induced IGFBP-4 protease activity. Thus, a decrease in IGFBP-4, which would make more IGF available to interact with its receptors and synergize with FSH to promote follicular growth and aromatization, appears to be a critical determinant of follicular selection for dominance.     

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)     

Identification of factors involved in target RNA-directed microRNA degradation

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.     

Persistence and recovery of regressing 3-mm ovarian follicles in heifers

The persistence and outcome of 3-mm follicles before the emergence of follicular wave 1 were studied every 6 hours in 15 heifers beginning on Day 14 (Day 0 = ovulation). A mean of 9.1 ± 1.3 persistent 3-mm follicles (P3Fs) per heifer was detected with persistence for 3.5 ± 0.1 days. The P3Fs either regressed continuously and remained in the 3-mm range (3.0–3.9 mm) or regressed but with a transient increase in diameter during regression. Some (43%) P3Fs were rescued to become growing follicles in wave 1. The number of follicles that became part of wave 1 was less (P < 0.0001) for follicles that originated from a P3F (4.2 ± 1.0 P3Fs) than for follicles that did not originate from a P3F (11.9 ± 1.6 follicles). The day of rescue of wave 1 follicles from a P3F (Day −1.1 ± 0.6) was earlier (P < 0.001) than for emergence of follicles at 3 mm that did not originate from a P3F (Day −0.5 ± 0.5). A cluster of 5.1 ± 0.6 P3Fs was identified in 10 of 15 heifers by the synchronized peaks of transient diameter increases at the 6-hour interval corresponding to Day −4.0 ± 0.3. Concentrations of FSH oscillated at 12-hour intervals with a peak (P < 0.05) 6 hours before and 6 hours after the beginning of a transient diameter increase during a P3F. Concentration of FSH was greater (P < 0.02) in heifers with a high number (11–18) of P3Fs per heifer (0.27 ± 0.02 ng/mL) than with a low number (2–9) per heifer (0.17 ± 0.008 ng/mL). Results supported the novel hypothesis that 3-mm follicles may persist for two or more days and may be rescued to become growing follicles of wave 1.     

冬虫夏草生境选择主导因子

采用样方法对冬虫夏草生境的海拔、坡度、植被和土壤等环境因子进行了探索研究。结果表明:(1)冬虫夏草主要分布在海拔4000～4500m、坡度为15°～30°的上坡位高山草甸中。(2)PCA分析表明:植被和地形是影响冬虫夏草分布的主导因子。(3)不同深度土壤养分含量统计分析表明:在不同深度层次的土壤中,水分、pH值、全氮和全磷的差异不显著,有机质、有机碳、水解氮和有效磷的差异极显著。(4)对土壤养分含量判别分析表明:5cm土壤中,吸湿水、有机碳、有机质和水解氮显著影响冬虫夏草种群分布;15cm土壤中,全氮显著影响冬虫夏草种群分布;25cm土壤中,吸湿水、有机碳和有机质显著影响冬虫夏草种群分布;而自由水、pH值、全磷和有效磷对冬虫夏草种群分布的影响不显著。(5)植物盖度、植物数量和坡度直接影响冬虫夏草种群分布,而土壤则通过影响植物和土壤温湿度来间接影响冬虫夏草种群分布。     

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.