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a Department of Dairy and Animal Science, The Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maturational (cytoplasmic and nuclear) and developmental (fertilization, pronuclei formation, and cleavage) competencies are influenced by the presence of follicular fluid (FF) and the size of the follicle from which porcine FF (pFF) is harvested. Although maturation of porcine oocytes in vitro (IVM) was inhibited during culture in pFF from small and medium-sized follicles [3], in later studies [4–8] the addition of pFF to the media improved maturation, penetration, and normal fertilization rates. However, polyspermic fertilization remains the major problem in porcine in vitro fertilization (IVF) [5, 6, 9]. Similar contradictory effects of FF have been reported for bovine oocytes [10, 11].
Follicle size plays an important role in oocyte maturation and embryonal development. Mature porcine oocytes seem to originate from preovulatory follicles; immature oocytes originate from smaller follicles [12]. Similarly, coculture of oocytes with follicular shells from large follicles significantly increased male pronucleus formation compared to coculture with shells from small follicles [13]. In cattle, oocytes from small follicles had significantly lower maturation and fertilization rates than those from medium and large follicles [14, 15].
Studies in pigs [16], cattle [17, 18], and sheep [19, 20] showed that oviductal epithelial cell coculture, or conditioned medium (CM) of oviductal tissue culture, promoted embryo development in vitro. This was apparently the effect of oviductal secretions, similar to those in vivo [21]. Brown and Cheng [22] reported that oviductal glycoproteins bind firmly to the zona pellucida of porcine oocytes, whereas Wegner and Killian [23] reported that a 97-kDa estrus-associated glycoprotein produced by the ampulla bound to and became localized in the periphery of the bovine zona pellucida. Oviductal glycoproteins have been found in the perivitelline space of mouse [24] and sheep [25, 26] oocytes and attached to the surface of sheep blastomeres [26], but the 97-kDa glycoprotein was in the perivitelline space of bovine oocytes or on bovine (7-day-old) blastomeres [23].
Thus, the presence of oviductal secretory products in the culture medium creates a unique microenvironment that reduces polyspermy and enhances fertilization and embryo development. This microenvironment might be enhanced by the presence of pFF, which is known to reduce polyspermy and increase fertilization rates.
We have hypothesized that pFF is an important factor in oocyte maturation and fertilization. The objective of the present studies, therefore, was to evaluate the effects of fresh or snap-frozen pFF, follicle size, and CM from oviducts on IVM and IVF of porcine oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collection of oocytes Ovaries were collected from prepubertal and cyclic gilts at a local abattoir, placed in saline (37°C) in an insulated container, and transferred to the laboratory. Follicles were classified according to size: small (3.0–5.0 mm), medium (5.1–7 mm), and large (over 7 mm). Their contents were aspirated with a 10-ml syringe fitted with an 18-gauge needle. Cumulus-oocyte complexes were washed three times in TALP-Hepes [27], and those with unexpanded cumulus layers were retained for culture.
Maturation medium The maturation medium (tissue culture medium [TCM]-199 with Earle's salts; Cellgro, Denver, CO) was modified [28, 29] and used as reported by Hagen et al. [30].
Preparation of supplements For experiments 1 and 2, pFF was aspirated from superficial small, medium, and large follicles of ovaries from prepubertal and cyclic gilts as described above. For experiment 3, pFF was aspirated from medium follicles only. Fluid was pooled within follicle-size category, centrifuged for 2 min (15 000 x g), snap frozen (dry ice and ethanol for 1–2 min), thawed, and then added to the medium (10%, v:v). Fetal calf serum (FCS; heat inactivated) was purchased from Gibco BRL (Grand Island, NY).
Culture of oocytes Drops (50 µl) of maturation medium were placed in sterile plastic culture dishes (60 x 15 mm, Falcon #1007; Becton, Dickinson and Co., Rutherford, NJ) and covered with paraffin oil (Baker, Phillipsburgh, NJ) equilibrated with PBS. Ten cumulus-oocyte complexes from each follicle size category were placed in each drop of prepared medium within 4 h after slaughter and incubated at 39°C in 5% CO2 for 48 h. At the end of incubation, oocytes were examined for cumulus cell expansion; they were then stripped free of cumulus cells with hyaluronidase (1 mg/ml; Sigma Chemical Co., St. Louis, MO) in TALP-Hepes under repetitive pipetting.
Preparation of spermatozoa Sperm-rich fractions (50 ml) from two ejaculates were obtained from a mature Yorkshire boar using the gloved-hand method. The fresh semen (20 ml/tube) was washed by centrifugation three times with warm sperm diluent (20 ml of saline supplemented with 10 mg BSA fraction V and 1 µg/ml gentamicin sulfate; Sigma). The first wash was at 250 x g for 3 min. Ten milliliters of the supernatant and 5 ml of the diluent were combined and then centrifuged for another 5 min at 500 x g. The supernatant was discarded, and fresh sperm diluent (15 ml) was added to the tube and centrifuged (500 x g) for another 5 min to remove seminal plasma. After washing, the pellets containing spermatozoa were resuspended to 6 x 107 cells/ml in TCM-199 supplemented with 10% FCS, gentamicin sulfate (1 µg/ml; Sigma), and 1 mg/ml caffeine (Sigma) for a total volume of 2 ml at pH 7.8. The sperm suspension was incubated for 60 min at 39°C in an atmosphere of 5% CO2 in air.
IVF Matured, stripped oocytes were transferred to drops of fertilization medium (50 µl, 10 oocytes per drop) consisting of a glucose-free Tyrode's-lactate solution supplemented with 6 mg/ml BSA (fatty acid-free fraction V; Sigma) and 2.2 mg/ml pyruvate. Ten microliters of diluted preincubated spermatozoa was added to each drop for a final sperm concentration of 107 cells/ml. Oocytes were cocultured with spermatozoa for 18 h at 39°C (5% CO2 in air).
Assessment of maturation and fertilization Stripped oocytes (all oocytes from experiment 1; 20% of the stripped oocytes from experiment 2) were mounted on glass slides, fixed [30], and stained with 1% orcein in water : acetic acid (60:40). Oocytes were observed under phase-contrast optics (x400, Olympus BH-2; Olympus Corp. of America, Lake Success, NY) to score the configuration of the chromatin and classified according to their meiotic stage. Oocytes in metaphase I, metaphase II, and diakinesis were scored as initiating or completing maturation [29]; those in germinal vesicle (GV) and germinal vesicle breakdown (GVBD) were considered immature. Degenerated oocytes were not included in the analysis.
At the end of the fertilization coculture, extraneous spermatozoa were washed from the oocytes by repetitive pipetting, and the oocytes were transferred into TALP-Hepes drops (50 µl) under paraffin oil and cultured for 6 h at 39°C in air. The oocytes were then fixed and stained as described above and were classified as penetrated, as having formed male pronucleus, as polyspermic, or as normally fertilized (syngamy completed).
Experiment 1. Effects of pFF and Follicle Size on Porcine IVM
Oocytes and pFF were aspirated from small, medium, and large follicles. On the day of use, maturation media were supplemented (10%, v:v) with either FCS (control group), fresh pFF, or snap-frozen pFF. Porcine FF treatments represented each of the follicle size categories already described, in a 3 x 7 factorial. The oocytes of each follicle size were treated with either FCS or separate pools of pFF (fresh or snap-frozen) from small and medium follicles.
Data were analyzed by the chi-square test and t-test (one sided) proportional analysis. A probability of p < 0.05 was considered statistically significant.
Experiment 2. Effects of pFF (Fresh or Snap-Frozen) and Follicle Size on Oocyte Maturation and Fertilization In Vitro
Media for maturation and fertilization were supplemented (10%, v:v) with either FCS (control group), fresh pFF, or snap-frozen pFF aspirated from medium-sized follicles and used for oocytes of each follicle size (control: 3 groups; treatment: 6 groups) in a 3 x 3 factorial design. Statistical analysis was as described for experiment 1.
Experiment 3. Effects of Oviductal CM on IVF of Porcine Oocytes
Ten Yorkshire gilts (7–9 mo) were observed for estrus daily and killed (The Pennsylvania State University, Meats Laboratory) either in the periovulatory stage (n = 5, Days 19–21 of the cycle) or in the midluteal stage (n = 5, Days 9–11 of the cycle). On each of five calendar dates, one gilt in each cycle stage was killed, for a total of five repetitions.
Oviducts were harvested and placed immediately in separate sterile conical centrifuge tubes (50 ml; Falcon) containing 20 ml of PBS and gentamicin sulfate (1 µg/ml; Sigma) and then transferred to the laboratory. Prior to processing, oviducts were washed (7 times) again with PBS containing gentamicin sulfate. They were then transferred to a sterile glass plate, trimmed of surrounding tissue, and cut longitudinally from the ampulla to the isthmus. Five pieces (3–4 mm) of the ampulla and five pieces (4–5 mm) of the isthmus, all proximal to the ampulla-isthmic junction, were dissected. Within 2 h of slaughter, the dissected pieces containing epithelium, stroma, and muscle layers were transferred to 100 x 15-mm Petri dishes (Quebec-grid #2070; VWR, Cleveland, OH) containing 15 ml of culture medium. The culture medium consisted of 15 ml serum-free RPMI 1640 (with L-glutamine and without NaHCO3; Sigma) and Dulbecco's Modified Eagle's medium (containing 1 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate; Gibco BRL), supplemented with 3 g/L D-glucose, 0.292 g/L L-glutamine, 2.85 g/L NaHCO3, 25 ng/ml epidermal growth factor, insulin-transferrin-sodium selenite (5 µg/ml, 5 µg/ml, and 5 ng/ml, respectively; Sigma), 1 µg/ml gentamicin sulfate, and 1% (v:v) antibiotic-antimycotic (Sigma). The oviductal explants were incubated for 48 h at 39°C in 5% CO2 in air. After the first 24 h of culture, the CM was replaced with fresh medium and explants were cultured for an additional 24 h. The wet weights of the oviductal explants were recorded at the end of culture. Conditioned culture medium from each cycle stage and day of culture was harvested and then centrifuged for 3 min (15 000 x g) to remove cellular debris before use as a supplement for the fertilization medium. Protein concentration of each oviductal CM sample was analyzed with the BCA (bicinchoninic acid) protein assay reagent (Pierce, Rockford, IL). The absorbance was read on a spectrophotometer (Microplate EL 311; Bio-Tek Instruments, Winooski, VT) at 562 nm.
The standard fertilization medium was supplemented (10%, v:v) with either FCS, pFF, luteal CM (LCM), periovulatory CM (PVCM), pFF and LCM (pFF+LCM, 10% of each, v:v), or pFF and PVCM (pFF+PVCM, 10% of each, v:v).
Data were analyzed by the Proc-Logist procedure [31]. There were five repetitions, six treatments, and four response variables (penetration, male pronucleus formation, polyspermy, and normal fertilization rates). The oviductal explant weights were analyzed with the t-test (one sided) proportional analysis; protein concentrations from the fresh and snap-frozen pFF and the oviductal CM samples were analyzed by the Wilcoxon rank sum test [32]. A probability of p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulus cells expanded in all treatments during culture. Snap-frozen pFF from small follicles improved (p < 0.05) maturation rates of oocytes from small follicles in comparison to those treated with FCS and fresh pFF. Similarly, maturation rates of small follicle-derived oocytes treated with snap-frozen pFF from medium follicles were higher than for those treated with fresh pFF from small follicles (p < 0.05, Table 1). Oocytes from medium-sized follicles exhibited higher maturation rates (p < 0.05) when treated with snap-frozen pFF from medium follicles than when treated with FCS and fresh pFF (Table 1). There was no difference (p > 0.05) among supplements in the maturation rates of oocytes aspirated from large follicles.
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Frozen pFF from small and medium follicles improved maturation rate only when it was used as a supplement for oocytes harvested from the same follicle category (Table 1). Again, maturation rate of oocytes from the large category was not affected (p > 0.05) by size of follicle from which pFF was collected. When data were pooled across oocyte sources, snap-frozen pFF from small follicles was found to have improved (p < 0.05) maturation rates over those seen with fresh pFF from small follicles (Table 1).
Oocytes from large follicles exhibited the highest maturation rates (p < 0.05; Table 1), regardless of supplement source. Similarly, oocytes from medium follicles had higher (p < 0.05) maturation rates than did those from small follicles (Table 1). The enhanced maturation rate associated with pFF from medium follicles resulted in its being used as the exclusive pFF supplement in subsequent experiments.
Experiment 2
Only 13 oocytes were recovered from large follicles; they were not included in the analysis. Cumulus cells in all treatments were well expanded after culture. The maturation rate of small follicle-derived oocytes treated with snap-frozen pFF from medium follicles was higher than for those treated with FCS (p < 0.05). However, overall maturation rates across either medium supplements or follicle size of origin did not differ (p > 0.05; Table 2).
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Penetration rates of oocytes from small follicles did not differ across treatments. Oocytes treated with snap-frozen pFF exhibited a higher rate (p < 0.05; Table 3) of male pronucleus formation. Lower (p < 0.05) polyspermy rates were observed for oocytes treated with pFF compared with those treated with FCS. The normal fertilization rate for oocytes treated with snap-frozen pFF was higher (p < 0.05) than for those treated with fresh pFF (Table 3).
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Rates of male pronucleus formation and normal fertilization of oocytes from medium follicles did not differ among treatments. However, oocytes treated with fresh pFF exhibited lower penetration rates (p < 0.05; Table 3) than FCS-treated oocytes, and oocytes treated with snap-frozen pFF had lower (p < 0.05) polyspermy rate than FCS-treated oocytes. Although polyspermy rate with the fresh pFF was double that with the snap-frozen pFF group, the difference was not significant (Table 3).
Experiment 3
There was no significant difference among response variables across repetitions (p > 0.05). Protein concentrations of the periovulatory-stage (2.85 mg/ml) and the luteal-stage (2.66 mg/ml) CM were similar (p > 0.05). Protein concentrations of fresh (36 mg/ml) and snap-frozen (35.4 mg/ml) pFF did not differ (p > 0.05). However, the oviductal explant wet weights from the periovulatory-stage (615.4 mg) and the luteal-stage (546.8 mg) oviducts were different (p < 0.05).
Oocyte maturation rates of small (77%) and medium (82%) follicles did not differ (p > 0.05).
Oocytes from small follicles The highest penetration rates were observed for oocytes treated with snap-frozen pFF or with pFF+PVCM (p < 0.05; Table 4). Oocytes treated with pFF+LCM exhibited the lowest (p < 0.05) rate of male pronucleus formation among all treatments. However, overall male pronucleus formation exceeded 73%.
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Lower (p < 0.05) polyspermy rates and higher normal fertilization rates were recorded for oocytes treated with snap-frozen pFF, PVCM, or pFF+PVCM, compared with FCS.
Oocytes from medium-sized follicles The highest penetration rate, highest male pronucleus formation rate, and lowest polyspermy rate were observed when either snap-frozen pFF or PVCM was added to the fertilization medium (p < 0.05; Table 4). Normal fertilization rates were lowest (p < 0.05) for oocytes treated with FCS in comparison to snap-frozen pFF, PVCM, pFF+PVCM, or pFF+LCM.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, pFF from small and large follicles inhibited maturation of porcine oocytes in vitro when the maturation medium was not supplemented with gonadotropins and steroids [3], apparently the result of an adverse effect on nuclear and cytoplasmic maturation [4, 28]. Bovine oocytes treated with bovine FF from small, medium, or large follicles had a lower rate of maturation than those treated with FCS [10], yet bovine FF treatment significantly increased maturation rates of bovine oocytes compared to controls [35]. Moreover, bovine FF from large follicles enhanced maturation and further development of bovine oocytes in vitro [36].
Thus, follicle size, an indicator of follicular development, influences the beneficial or inhibitory properties of the FF in terms of developmental capacity of the oocyte. The highest maturation rates were achieved with oocytes from large follicles, regardless of treatment, supporting the report of Hunter and Wiesak [12] that oocytes in advanced nuclear maturation stages after the LH surge were from large follicles; less-mature oocytes originated from smaller follicles. Similarly, bovine oocytes from medium and large follicles exhibited higher rates of maturation, penetration, male pronucleus formation, and blastocyst formation compared to those from smaller follicles [14]. Blastocyst formation was greater for oocytes harvested from follicles more than 6 mm in diameter compared to those from follicles less than 6 mm [11, 15]. Thus, follicular heterogeneity results in asynchrony of oocyte maturation (in vivo or in vitro), which in turn may determine the developmental capacity of the oocyte and the viability of the early embryo.
In experiment 2, snap-frozen pFF enhanced rates of male pronucleus formation and fertilization and decreased polyspermy rate of oocytes from both small and medium follicles. It is postulated that the snap-freezing process of pFF liberates or modifies and activates one or more specific unknown factors that enhance fertilizability of porcine oocytes in vitro.
Supplementation of maturation medium with either untreated, heated, or defatted pFF promotes cumulus expansion and maturation rates of porcine oocytes compared to those treated with FCS, but does not affect rates of fertilization, polyspermy, or male pronucleus formation [6]. Fractionating pFF reduced polyspermy and increased fertilization rate compared to that in controls [6]. In contrast, supplemental pFF in the prefertilization and fertilization media of porcine oocytes resulted in a significant reduction in polyspermic fertilization [7]. However, in a later study, the inclusion of pFF in the maturation medium enhanced oocyte maturation and male pronucleus formation but failed to reduce polyspermy [8].
Earlier studies suggested that pFF from immature follicles contains a small (< 10 kDa) polypeptide that maintains oocytes in the dictyate stage of the first meiotic prophase [37, 38], while more recent studies indicate the opposite [4–6]. The reasons for this disparity are unclear. In the present study, snap-frozen pFF in the maturation and fertilization media increased rates of maturation and normal fertilization and reduced polyspermy. Yoshida et al. [6] suggested that one or more heat-labile (56°C) acidic factors in pFF with a molecular mass between 10 and 200 kDa are responsible for oocyte maturation. Further, the lower polyspermy rates observed after addition of the highly purified fractions suggest that the same factor(s) plays a role in reducing polyspermy. In contrast, Daen et al. [39] suggested that the pFF factor has a molecular mass of < 6.5 kDa and is heat stable at 100°C for 15 min. However, pFF may not be the only source of this factor(s). A "cumulus-expansion enabling" factor with protein-like properties is secreted by mouse oocytes in vitro [40]. A similar, non-species-specific cumulus expansion-stimulating factor(s) is present in pig, cow, rabbit, and rat serum [40]. The active factor(s) in pig serum is heat stable at 100°C for 30 min and has an estimated molecular mass between 1 and 6.5 kDa, but it does not enhance the formation of male pronucleus of serum-treated porcine oocytes (second metaphase stage) in vitro [40].
Oocytes treated with snap-frozen pFF, PVCM, or the combination of the two may have a higher probability for further embryo development than those treated with FCS, due to less polyspermy and a higher rate of fertilization. This is consistent with results of Nagai and Moor [41], who reduced polyspermy from 95% to 58% by using oviductal epithelial cells in the fertilization medium, and with those of Kano et al. [42], whose coculture system with oviductal epithelial cells or CM of oviductal epithelial cells reduced polyspermy without reducing rate of fertilization: although polyspermy was reduced by epithelial cells and CM, normal fertilization rates were maintained. These results suggest that macromolecules secreted by the oviductal cells interact with the gametes, leading to a more effective block to polyspermy and enhanced fertilization. However, pFF was not included in those coculture systems, and the oviductal epithelial cells or the oviductal CM was obtained either from estrual [41] or prepubertal gilts [42].
The stage of the cycle, or the physiological and hormonal status of the animal from which the oviducts used in coculture are obtained, may have an influence on the gametes or on further embryo development. An estrus-associated oviductal fluid glycoprotein was reported in sheep [26, 43] and cattle [23, 44], and two estrus-associated oviductal fluid glycoproteins were found in pigs [22]. These glycoproteins vary in molecular weight, but they share a common feature: all are present in the oviductal fluid during estrus and not during the luteal phase [22, 23, 43]. These glycoproteins may facilitate fertilization or further embryo development [23, 44, 45], and might have been operative in the present studies. Conversely, Eyestone et al. [46] found no effect of stage of cycle on the response of early bovine embryos to oviduct tissue-conditioned medium.
Although glycoproteins were not quantified in the present study, the results presented here are consistent with a protective role of the estrus-associated glycoproteins [47]. The glycoproteins bound to the zona pellucida may provide a protective shield around the oocyte and the early embryo by participating in the zona reaction and blocking polyspermy. However, the mechanism of action of these macromolecules has not been elucidated.
In the present study, no synergism between pFF and oviductal CM was observed. Although we are aware of no fertilization coculture system that utilizes both FF and oviductal CM, embryonic development is supported by coculture of oocytes and sperm from sheep [19, 20, 44], cattle [17, 48], and pigs [16, 41] in the presence of oviductal epithelial cells or oviductal CM obtained from animals at estrus. Only Kano et al. [42] found a reduction in polyspermy from using oviductal epithelial cells or oviductal CM obtained from prepubertal gilts. Thus, during the peri- and postovulatory stages, the oviducts of several species secrete specific molecules that enhance embryonic development by providing the necessary factors (embryotrophic) for normal embryo development in vitro or inactivating potentially detrimental substances in the culture medium.
We are aware of no other study that has examined the effects of luteal-stage oviductal epithelial cells or oviduct CM on oocyte maturation and fertilization in vitro. In the present study, the combination of snap-frozen pFF and LCM—but not PVCM—increased polyspermy and decreased normal fertilization rates compared to those with snap-frozen pFF alone. Thus, LCM may have an adverse effect on oocyte fertilizability. There are at least two possible explanations for these adverse effects. First, the putative estrus-related glycoproteins that enhance gamete interaction and normal embryo development [21] may be absent. Their synthesis is regulated by estradiol-17ß; they are released by progesterone [49]. Under progesterone domination, the oviductal secretory cells—which are responsible for the synthesis of the glycoproteins—lose the receptors for estradiol-17ß, the primary stimulus for the initiation of protein synthesis [45], and the glycoproteins are not synthesized. Secondly, elevated progesterone during the luteal phase may impair normal fertilization by increasing the rate of polyspermic fertilization. Administration of progesterone prior to ovulation significantly increases the incidence of polyspermy and accelerates passage of oocytes through the oviducts into the uterus [50]. Local administration of progesterone in the oviductal wall, near the uterotubal junction, increased polyspermy [51].
Consequently, if putative beneficial oviductal glycoprotein(s) are not present in LCM and elevated progesterone levels in the oviductal fluid have an adverse effect on fertilization, they could neutralize the beneficial effects of the snap-frozen pFF.
In conclusion, pFF has beneficial effects on porcine oocyte maturation and developmental capacity in vitro. Snap-freezing of pFF may modify and induce specific factors that play a positive role or inhibit factors that play a negative role in IVF. However, further studies are needed to identify the mechanisms of action of these inhibitory or promoting factors of pFF on oocytes in order to establish an effective porcine IVM and IVF system. Supplementation of the fertilization medium with either pFF or oviductal CM of periovulatory gilts reduces polyspermy and enhances normal fertilization in vitro. Luteal-stage oviduct CM, when used as a supplement for fertilization medium—whether alone or with pFF—reduces porcine oocyte fertilizability in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D.R. Hagen, Department of Dairy and Animal Science, The Pennsylvania State University, 324 Henning Building, University Park, PA 16802. FAX: 814 863 6042; dhagen{at}das.psu.edu
Accepted: August 18, 1998.
Received: July 21, 1997.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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