01 Feb

Dynamic Changes in Meiotic Progression: RESULTS

RESULTSExperiment 1

In the pig, MI is a relatively long component of the meiotic transition from GV to MII compared with other phases (the diakinesis of prophase I, prometaphase I, AI, and TI). Therefore, GV, MI, and MII were used as representative stages to indicate meiotic progression. As shown in Figures 1 and 2, more than 80% of oocytes from both gilts and sows remained at the GV stage after 44-48 h of culture in medium B, and less than 10% reached MII. Medium supplementation with LH had no effect (P > 0.05) on the maturation of oocytes from either gilts or sows. Significantly higher proportions of oocytes matured to MII in media F and E in both gilts (50.0% ± 4.2% and 54.8% ± 4.3%, respectively; P < 0.001) and sows (87.6% ± 3.4% and 78.8% ± 3.9%, respectively; P < 0.001) compared with those in the basic medium with or without LH. The rate of maturation of gilt oocytes to MII in media F and E was significantly lower than that of sow oocytes (P < 0.001); significantly more gilt oocytes (FSH, 23.4% ± 3.9%; EGF, 34.8% ± 4.6%) than sow oocytes (FSH, 4.1% ± 2.1%; EGF, 4.4% ± 2.0%; P < 0.05) stayed at GV. Combined supplementation with FSH and EGF resulted in an additive effect on nuclear maturation in gilt oocytes (93.7% ± 2.1%), which was comparable to that in sow oocytes (93.1% ± 2.8%). Addition of LH to medium containing either FSH or EGF (or both) had no further effect on the rates of nuclear maturation (Figs. 1 and 2). canadian neighbor pharmacy

Experiment 2

In our previous studies using CHX, LH alone was used to supplement a basic maturation medium similar to the present one (0.57 mM cysteine added, but without any effect on nuclear maturation; data not shown), and mei-otic progression was synchronized and highly predicable. In particular, almost all (>80%) meiotically competent oocytes reached MI by 14 h after removal of CHX, with none entering AI/TI; this proportion was maximal and comparable to that of oocytes that reached MII by 24 h. Therefore, in the present study, 14 and 24 h after CHX pretreatment were used as the key time points for assessing meiotic progression.

No significant difference was observed between gilt and sow oocytes in the rates of CHX-synchronized nuclear maturation in medium B (55.2% ± 5.6% and 58.8% ± 3.3%, respectively; not significant [NS]) or medium E (86.6% ± 4.0% and 86.7% ± 7.8%, respectively; NS). Moreover, in contrast to conventional culture (experiment 1), pretreatment with CHX caused almost all oocytes to undergo GVBD (84-100%) regardless of culture media or oocyte origin, although the proportion of oocytes maturing to Mil at 24 h was relatively low in the basal medium (P < 0.05). Therefore, data from gilt and sow oocytes (cultured separately) were pooled for further examination of meiotic progression (variances within pooled data remained low). As shown in Figures 3 and 4, the majority of oocytes had matured to MI and MII at 14 and 24 h of culture after CHX pretreatment, respectively, in media B, L, E, and LE. At 14 h, the rates of maturation to MI were similar in all media (78.8% ± 4.6%, 85.6% ± 3.6%, 88.9% ± 3.5%, and 83.8% ± 4.2% in media B, L, E, and LE, respectively; NS). In medium supplemented with FSH, a significantly lower proportion of oocytes matured to Mi (43.8% ± 5.3%, P < 0.001), with a correspondingly higher proportion remaining at the GV stage (39.3% ± 5.9%, P < 0.001) until 14 h; only 48.4% of oocytes (P < 0.001) had reached MII by 24 h. However, a 12-h extension of culture (until 36 h) in medium F allowed the majority of oocytes to mature to MII (70.5% ± 2.9%) (Fig. 5). Addition of LH, EGF, or both to maturation medium in the presence of FSH had no significant effect on meiotic progression (Figs. 3-5).

Experiment 3

This experiment investigated whether CHX pretreatment and alteration of the speed of nuclear maturation by FSH would affect early embryo development after fertilization. To obtain similar nuclear maturation rates between control and treated oocytes before IVF, medium LE was chosen as the maturation medium, because neither medium B nor medium L stimulated maturation. As in most previous studies, LH was added, because it may provide general support for cytoplasmic maturation despite its lack of effect on nuclear maturation (experiment 2). Whereas the oocytes cultured without FSH were from sows, the three replicate batches cultured with FSH represented both gilts and sows, with at least one coming from gilts; variance between the batches in each treatment remained low (Fig. 6). As shown in Figure 6, the rates of normal cleavage 2 days after fertilization of CHX-pretreated oocytes were similar to those in untreated counterparts when matured with (71.3% ± 2.9% and 75.3% ± 3.1%, respectively; NS) or without (37.7% ± 3.0% and 43.0% ± 2.9%, respectively; NS) FSH. However, the frequencies of oocytes/zygotes developing to the blastocyst stage by Day 6 were significantly higher following CHX pretreatment than in untreated groups, both with (32.8% ± 2.0% and 10.3% ± 1.5%, respectively; P < 0.001) and without (16.7% ± 1.5% and 9.4% ± 1.2%, respectively; P < 0.001) FSH. Accordingly, a significantly higher proportion of Day 2-cleaved embryos from CHX-pretreated oocytes had developed to the blastocyst stage by Day 6 compared with untreated counterparts, both with (46.8% ± 4.9% and 14.3% ± 2.5%, respectively; P < 0.001) and without (44.7% ± 2.9% and 22.3% ± 1.4%, respectively; P < 0.001) FSH (data not shown in Fig. 6).

Supplementation of the maturation medium with FSH in the presence of LH and EGF significantly increased the rate of cleavage (P < 0.001) in both CHX-pretreated and untreated oocytes. Moreover, a positive interaction was observed between CHX pretreatment and FSH, leading to a substantially improved rate of development to the blastocyst stage (32.8% ± 2.0%) when compared to the effect of each individually (10.3% ± 1.5% and 16.7% ± 1.5%, respectively; P < 0.05).

Figure 7 shows a representative group of embryos derived from CHX-pretreated gilt oocytes matured in the presence of FSH that had developed to the blastocyst stage at Day 6 after IVF. The cell count for all treatments was between 50 and 65 cells/embryo at Day 6 (NS). There appeared to be no difference between gilts and sows in the development of oocytes pretreated with CHX to the blastocyst stage after IVF. As shown in Table 1, the use of a variety of IVF protocols (variables are not specifically indicated but include IVF procedure and period of oocyte-sperm coincubation) resulted in a wide range (P < 0.001) of penetration and polyspermy rates in both CHX-pretreat-ed and control oocytes. However, no difference from control was observed in either mean penetration rate (34.0% ± 1.6%; control, 29.5% ± 1.6%; NS) or mean polyspermy rate (14.3% ± 1.8%; control, 13.2% ± 1.9%; NS). The proportions of spontaneously parthenogenetically activated oocytes following overmaturation or with sperm-free IVF were very low (<5%), with no difference found between treatments.
Fig1Dynamic Changes in Meiotic Progression-1
FIG. 1. Effect of hormones on gilt oocyte maturation: proportion of oocytes at the GV, MI, and MII stages following conventional culture for 44-48 h in basal medium (B) or medium supplemented with LH (L), EGF (E), or FSH (F), either alone or in combinations, examined under a phase-contrast microscope at X400 magnification. Error bars represent the approximate SEMs estimated from a linear model fitted assuming binomial errors. Both EGF and FSH had statistically significant (P < 0.001) effects on the proportion of oocytes at the GV and MII stages.

Fig2Dynamic Changes in Meiotic Progression-2
FIG. 2. Effect of hormones on sow oocyte maturation: proportion of oocytes at the GV, MI, and MII stages following conventional culture for 44-48 h in basal medium (B) or medium supplemented with LH (L), EGF (E), or FSH (F), either alone or in combinations, examined under a phase-contrast microscope at X400 magnification. Error bars represent the approximate SEMs estimated from a linear model fitted assuming binomial errors. Both EGF and FSH had statistically significant (P < 0.001) effects on the proportion of oocytes at the GV and MII stages.

Fig3Dynamic Changes in Meiotic Progression-3
FIG. 3. Effect of hormones and CHX on porcine oocyte maturation: proportion of oocytes (from gilts and sows) at the GV, MI, and MII stages following culture for 14 h in basal medium (B) or medium supplemented with LH (L), EGF (E), or FSH (F), either alone or in combinations, preceded by preincubation with CHX for 12 h in the same medium and examined under a phase-contrast microscope at X400 magnification. Error bars represent the approximate SEMs estimated from a linear model fitted assuming binomial errors. The FSH had statistically significant (P < 0.001) effects on the proportion of oocytes at the GV and MI stages.

Fig4Dynamic Changes in Meiotic Progression-4
FIG. 4. Effect of hormones and CHX on porcine oocyte maturation: proportion of oocytes (from gilts and sows) at the GV, MI, and MII stages following culture for 24 h in basal medium (B) or medium supplemented with LH (L), EGF (E), or FSH (F), either alone or in combinations, preceded by preincubation with CHX for 12 h in the same medium and examined under a phase-contrast microscope at X400 magnification. Error bars represent the approximate SEMs estimated from a linear model fitted assuming binomial errors. Basal and FSH-supplemented media had statistically significant (P < 0.05) effects on the proportion of oocytes at the MII stage.

Fig5Dynamic Changes in Meiotic Progression-6
FIG. 5. Effect of hormones and CHX on porc ine oocyte maturat i on: pro-porti on of oocytes (from g ilts and sows) at the GV, MI, and MII stages following culture for 36 h in medium supplemented with LH (L) and/or EGF (E) in the presence of FSH (F) preceded by preincubation with CHX for 12 h in the same medium and examined under a phase-contrast microscope at X400 magnification. The error bars represent the approximate SEMs estimated from the linear model fitted assuming binomial errors. The EGF had statistically significant (P < 0.05) effects on the proportion of oocytes at the MII stage.

Fig6Dynamic Changes in Meiotic Progression-5
FIG. 6. Cleavage and blastocyst formation rates of oocytes from gilts and sows matured conventionally or after preincubation with CHX for 12 h with or without FSH in medium LE and then fertilized and cultured for 6 days. Error bars represent the approximate SEMs estimated from a linear model fitted assuming binomial errors. The CHX had a statistically significant (P < 0.001) effect on blastocyst rate at 6 days. The FSH had a statistically significant (P < 0.001) effect on both cleavage rate at 2 days and blastocyst rate at 6 days and interacted significantly (P < 0.05) with CHX for blastocyst rate at 6 days.

Fig7Dynamic Changes in Meiotic Progression-7
FIG. 7. Examples of Day 6 embryos derived from gilt oocytes matured after preincubation with CHX for 12 h with FSH in medium LE. A dish of 30 oocytes was cultured and fertilized (photograph taken at 5.5 days and X50 magnification). Arrows indicate blastocysts.

TABLE 1. Effect of CHX pretreatment before maturation culture on fertilzation parameters of pig oocytes.a
table1Dynamic Changes in Meiotic Progression-8
a Gilt or sow oocytes were matured with or without CHX pretreatment and fertilized using various modifications (protocols 1-7) of the IVF procedure described in Materials and Methods. Mean percentage and approximate SEMs are predicted from a linear model fitted assuming binomial errors.

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