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Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Departments of ¹ Veterinary Physiology and Pharmacology and ² Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4466, USA

Correspondence should be addressed to Katrin Hinrichs; Email: khinrichs{at}cvm.tamu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Research on developmental competence of in vitro matured equine oocytes has been scant, largely due to low availability of oocytes and lack of efficient techniques for in vitro fertilization (IVF) (Squires 1996, Hinrichs 1998). Recently, techniques for intracytoplasmic sperm injection (ICSI) using the piezo drill have been developed in the horse, which have resulted in high fertilization and cleavage rates (Choi et al. 2002a, Galli et al. 2002a). Several laboratories have produced equine blastocysts by in vitro culture (IVC) of ICSI zygotes (Dell’Aquila et al. 1997a; Guignot et al. 1998, Li et al. 2001, Galli et al. 2002a, Choi et al. 2003a), but the proportion of embryos reaching the blastocyst stage in vitro has typically been less than 10%. The limited success of in vitro embryo production in the horse may be related to the low developmental competence of the oocytes used; however, few studies have been performed to evaluate factors affecting developmental competence of equine oocytes.

Methods for classification of recovered equine oocytes vary among laboratories. Many laboratories have selected oocytes with compact cumuli (Cp) for culture and discarded oocytes with expanded cumuli (Ex) (Fulka & Okolski 1981, Willis et al. 1991, Choi et al. 1993, Goudet et al. 1998, Li et al. 2000, Tremoleda et al. 2001), as is done in most other species. However, nuclear maturation rates of Ex equine oocytes have been shown to be similar to (Zhang et al. 1989, Hinrichs et al. 1993, Dell’Aquila et al. 2001), or higher than (Hinrichs et al. 1995, Alm & Hinrichs 1996, Hinrichs & Williams 1997, Hinrichs & Schmidt 2000, Choi et al. 2002b), those for Cp oocytes. Information on the developmental competence of these two types of oocytes is limited. Compact oocytes had a reduced ability to respond to activation stimuli (Hinrichs et al. 1995), and reduced ability to form a male pronucleus after ICSI (Dell’Aquila et al. 1997b) when compared to Ex oocytes. After ICSI, Cp oocytes have been reported to have lower (Hinrichs et al. 2002: duration of maturation 24 h) or equal (Franz et al. 2003: duration of maturation 30 h) rates of cleavage when compared to Ex oocytes.

The lower meiotic, and possibly developmental, competence of Cp oocytes may be related to their follicular origin. Compact oocytes are recovered from viable follicles, whereas Ex oocytes are recovered from atretic follicles (Hinrichs & Williams 1997). Viable follicles appear to keep the enclosed oocyte in a more juvenile state; in contrast, atretic follicles allow prematurational changes, such as chromatin condensation, to occur within the oocyte (Hinrichs & Williams 1997). Compact oocytes take longer to reach metaphase II than do Ex oocytes (Zhang et al. 1989, Hinrichs et al. 1993, Alm & Hinrichs 1996, Choi et al. 2002b). The duration of in vitro maturation (IVM) that affords maximum developmental competence for equine oocytes is unknown; it is possible that this may vary according to oocyte type. This possibility is supported by the finding that culture in the presence of the meiosis inhibitor, roscovitine, for 24 h before starting culture for IVM increased the developmental competence of Cp, but not of Ex, equine oocytes (Franz et al. 2003).

Assessment of developmental competence is dependent on having a culture system that supports embryo development; however, little work has been done on requirements for culture of early equine embryos. We previously found that development at 96 h was similar for embryos cultured in G1.2 or in a modified CZB medium (Choi et al. 2003b), establishing the possibility of using CZB as a base medium for culture of early equine embryos. One of the most influential components of embryo culture media is glucose, which can have an inhibitory effect on development of preimplantation embryos depending on stage, species and medium composition (review, Bavister 1995). There is only one previous report on the effect of glucose concentration on development of equine embryos: Azuma et al.(1995) investigated glucose concentrations of 0.5 and 5.5 mM for early equine embryo culture, and found that culture in the low concentration on days 1–4, followed by increasing glucose concentration, improved development to the morula stage at day 8.

Procedures performed during ICSI may also affect subsequent embryonic development. In the first report of use of a piezo drill for ICSI, performed in mice, it was suggested that oocytes should be manipulated at low temperatures and held briefly before culture to prevent membrane lysis (Kimura & Yanagimachi 1995). This group later performed ICSI at room temperature (23–25 °C) without a holding period and obtained similar survival rates (Wakayama & Yanagimachi 1998a,b). In the horse, some researchers use a heated stage for conventional ICSI (Dell’Aquila et al. 1997a, b, 2001, Cochran et al. 1998), but there is no information available on the effect of manipulation temperature on the survival or early development of equine embryos.

The aim of this study was to evaluate differences in developmental competence between Ex and Cp oocytes, and the effect of different durations of oocyte maturation on their developmental competence, by studying embryo development after ICSI. Because methods for culture of later-stage equine embryos have not yet been optimized, embryo development was assessed at 48 h after fertilization to minimize the effect of culture conditions on embryo performance. In addition, we evaluated the effects of manipulation temperature (room temperature vs 37 °C) and two glucose concentrations (0.55 vs 5.5 mM) during in vitro culture after ICSI of Ex and Cp oocytes on development at 96-h culture.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experiment 1: competence of Ex and Cp oocytes after different periods of maturation
Oocyte collection
This study was conducted in the months of November to June. Ovaries were obtained from two abattoirs and transported to the laboratory at room temperature (26–34 °C, 3–4-h transport time). Adnexa were trimmed from the ovaries with scissors, and the ovaries were cleaned with sterilized gauze. All visible follicles were opened with a scalpel blade, and the granulosa layer of each follicle was scraped using a 0.5 cm bone curette. The contents of the curette were washed into individual petri dishes with Hepes-buffered TCM199 with Hanks’ salts (Gibco Life Technologies, Inc., Grand Island, NY, USA) plus ticarcillin (0.1 mg/ml, SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA). The contents of the petri dishes were examined using a dissection microscope at x 10–20 magnification. Oocyte–cumulus complexes were classified as compact, expanded or degenerating depending on the expansion of both mural granulosa and cumulus, as described previously (Hinrichs & Williams 1997, Hinrichs & Schmidt 2000). Oocytes with any sign of expansion of either the cumulus or the mural granulosa (from having individual cells visibly protruding from the surface to having full expansion with copious matrix visible between cells) led to the classification of Ex. Oocytes having both compact cumulus and compact mural granulosa were classified as Cp. Oocytes showing signs of degeneration (shrunken, dense or fragmented cytoplasm) were discarded.

In vitro maturation
The oocytes were washed twice in maturation medium (TCM199 with Earle’s salts (Gibco), 5 µU/ml follicle-stimulating hormone (FSH) (Sioux Biochemical, Inc., Sioux Center, IA, USA), 10% fetal bovine serum (FBS) (Gibco) and 25 µg/ml gentamycin (Gibco)), and then cultured in droplets of this medium at a ratio of 10 µl medium per oocyte, under light white mineral oil (Sigma Chemical Co., St Louis, MO, USA) at 38.2 °C in 5% CO₂ in air for 24, 30 or 42 h. After maturation, oocytes were denuded of cumulus by pipetting in a solution of 0.05% hyaluronidase in TCM199 with 5% FBS. Denuded oocytes were selected for presence of a polar body. Oocytes not exhibiting a polar body were fixed in buffered formol saline, mounted on a slide with 6.5 µl of 9:1 glycerol:PBS containing 2.5 µg/ml Hoechst 33258, and examined using fluorescence microscopy to determine the chromatin configuration.

Sperm preparation
Frozen equine semen was used for ICSI. Semen straws were thawed at 37 °C for 30 s, and 200 µl of the semen was placed on the bottom of a 5 ml tube containing 1 ml of modified Tyrode’s albumin lactate pyruvate (Sp-TALP) (Parrish et al. 1988) for swim-up. After 20-min incubation at 38.2 °C in an atmosphere of 5% CO₂ in air, 0.6 ml of medium was collected from the top of the tube, and centrifuged at 327 g for 3 min in a 1.7 ml polypropylene tube. The sperm pellet was resuspended and washed once with the same medium. The supernatant was removed and the pellet was used to provide sperm for ICSI.

Intracytoplasmic sperm injection
The ICSI procedure was conducted as previously described (Choi et al. 2002a) using a piezo drill. Briefly, the outside diameter of the sperm injection pipette was 7–8 µm. A 120–140 µm (outside diameter) pipette was used to hold the oocytes. Immediately before injection, 1 µl of sperm suspension was placed in 3 µl of Sp-TALP containing 10% PVP (Sigma) under oil. Sperm injection was carried out in a separate 50 µl drop of Hepes-buffered TCM199 containing 10% FBS. Each spermatozoon was immobilized by applying a few pulses with the Piezo drill (Burleigh Instruments, Inc., Fishers, NY, USA) to the sperm tail immediately before injection. All manipulations were performed at room temperature. Injected oocytes were held for 20 min at room temperature in the same medium to heal the broken membrane slowly.

In vitro culture
After ICSI, fertilized oocytes were washed twice in G1.2 medium (G1.2/G2.2, IVF Science, Denver, CO, USA) and cultured in the same medium at 38.2 °C under 5% CO₂ in air. At 48 h post-ICSI, all oocytes/embryos were fixed and stained to examine the number and status of nuclei. Only nuclei that appeared to be normal were included in the embryo nucleus number; nuclei showing signs of degeneration (vacuolization, condensation or fragmentation) were disregarded.

Experiment 2: effects of manipulation temperature and glucose concentration
This study was conducted in the months of April to August. Media used for holding oocytes, swim-up for sperm, manipulation and culture were CZB-H, Sp-CZB, CZB-M and CZB-C respectively, which were modified from CZB (Chatot et al. 1989, Choi et al. 2003b). The osmolarity of these media was 298 ± 3 mOsm.

Oocytes were recovered and matured as described above; however, based on results of experiment 1, Ex and Cp oocytes were denuded after 24–25-h IVM and held in CZB-H until 30-h IVM. Sperm swim-up for ICSI was performed as described above, but using Sp-CZB for sperm preparation instead of Sp-TALP. Sperm were injected into oocytes in a microdroplet of CZB-M either at room temperature or at 37 °C. For the 37 °C treatment, a heating stage (Fryer A-50; Fryer Co., Carpentersville, IL, USA) was used. Oocytes manipulated at 37 °C were immediately transferred to CZB-H and incubated at 38.2 °C; oocytes injected at room temperature were held for 20 min at room temperature in a separate drop of manipulation medium before being placed in culture. The presumptive zygotes were cultured in CZB-C with either 0.55 mM glucose or 5.5 mM glucose at 38.2 °C in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂. After 96-h culture, without a change of medium, embryos were fixed and stained as described above to examine the number and status of nuclei.

Statistical analysis
Differences in maturation rates, fertilization rates, morphologic and normal cleavage rates, and proportions of embryos developing to the different stages among groups were evaluated using chi-square analysis, with Fisher’s exact test being used when the expected value for any parameter was less than 5. Differences in average nucleus number among maturation durations, glucose concentrations and manipulation temperatures, and between cumulus morphologies, were evaluated using analysis of variance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
A total of 1198 ovaries were processed, and 6861 follicles were scraped, for an average of 5.7 follicles per ovary. Of recovered oocytes, 1139 were Cp, 2514 were Ex and 318 were degenerating. For this study, 1165 Ex and 1103 Cp oocytes were used, and the remainder were used on a separate project.

Experiment 1
Of the 500 Ex and 485 Cp oocytes cultured for IVM, 12 Ex and 6 Cp oocytes were broken during denuding of cumulus cells. After denuding, 319 Ex (65%) and 114 Cp (24%) oocytes were selected for having a first polar body. Of 178 Ex and 365 Cp oocytes without visible polar bodies, 10 (2% of total oocytes) Ex and 24 (5% of total oocytes) Cp were found to be in metaphase I (MI) on fixation and staining, and 6 (3%) Ex and 1 (0.3%) Cp were in metaphase II (MII). Maturation rates to MI and MII (visible polar body plus oocytes in MII on staining) in the different treatments are presented in Table 1. Maturation rates of Ex oocytes increased significantly from 24 to 30 h. The maturation rates for Ex oocytes were significantly (P < 0.001) higher than those for Cp oocytes at each time period. The proportion of Cp oocytes in MI at 24 h was significantly (P < 0.05) higher than that for 42 h.

A total of 402 oocytes with a polar body were manipulated for ICSI, and 394 (98%) were successfully injected. The rates of morphologic cleavage and cleavage with normal nuclei after ICSI in the different treatments are shown in Table 2. For Ex oocytes, the rate of normal cleavage (more than two nuclei) was significantly higher in the 24-h than in the 30- or 42-h treatments. For Cp oocytes, the morphologic cleavage rate was significantly higher (P < 0.05) in the 30-h than in the 24- or 42-h treatments. When Ex and Cp oocytes were compared within treatments, Ex oocytes had a higher rate of normal cleavage than did Cp oocytes when oocytes were matured for 24 h or 42 h, but there was no significant difference in normal cleavage between Ex and Cp oocytes when matured for 30 h.

View larger version (76K): [in this window] [in a new window]   Figure 1 A fertilized six-cell equine embryo originating from an oocyte with compact (Cp) cumulus and cultured for 48 h in G1.2 medium after intracytoplasmic sperm injection as visualized by (a) bright field and (b) fluorescence and bright-field microscopy after Hoechst 33258 staining (six nuclei). Scale bar represents 50 µm.

View larger version (94K): [in this window] [in a new window]   Figure 2 Three embryos originating from oocytes with compact (Cp) cumulus, fertilized by intracytoplasmic sperm injection at room temperature and cultured in CZB-C with 5.5 mM glucose for 96 h in vitro, showing 24, 30 and 8 nuclei including mitotic figures (arrows). Bar represents 50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The increased meiotic competence of Ex oocytes is likely to be related to changes undergone by the oocyte during early follicle atresia (Hinrichs & Williams 1997). Our results suggest that these changes may also affect the developmental competence of Ex oocytes. The proportion of Ex oocytes cleaving normally in this study was significantly higher for oocytes matured 24 h than for those matured 30 or 42 h (Table 2); in contrast, Cp oocytes matured for 30 h had a significantly higher rate of morphologic cleavage than did the other two maturation times, and also the highest rate of normal cleavage among the three maturation time periods.

While Ex oocytes at 24 h had higher rates of normal cleavage than did Cp oocytes at any time period (Table 2), the extent of embryonic development achieved was greater for Cp than for Ex oocytes in both experiment 1 and experiment 2. Because Ex oocytes undergo prematurational changes within the follicle, they may be ‘aged’ relative to Cp oocytes and thus may be more easily activated, as has been shown for in vitro aged oocytes in cattle (Presicce & Yang 1994). This may be reflected in the increased rate of cleavage after ICSI seen in Ex oocytes in the present study. However, Ex oocytes may have deficiencies in cytoplasmic competence related to follicle degeneration and/or oocyte aging, which would explain the relatively high proportion of embryos having delayed development (experiments 1 and 2). In cattle, oocytes with pronounced cumulus expansion had decreased developmental competence (Blondin & Sirard 1995).

The medium used for oocyte maturation in this study is relatively simple (M199 with FSH and FBS). Maturation medium may affect developmental competence; however, in the horse, no difference in rates of nuclear maturation, cleavage, or development to compact morulae or blastocysts has been found between relatively simple media and those supplemented with epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, and/or additional hormones (luteinizing hormone (LH), E₂ and P₄) (Galli et al. 2002b, Choi et al. 2003b).

High glucose concentration (5.5 mM) has been reported to be detrimental to the development of equine embryos produced by fertilization of zona-dissected oocytes (Azuma et al. 1995). However, in the present study, high glucose did not inhibit development of ICSI embryos from Ex or Cp oocytes. Instead, the development of embryos from Cp oocytes was improved by high glucose, as reflected in significantly higher morphologic cleavage rates in this group. In addition, the rate of normal cleavage and the average nucleus number for Cp oocytes in the 5.5 mM glucose treatment were not depressed, being higher (though not significantly so) than the corresponding rates for Cp oocytes cultured in the low-glucose treatment. The average number of nuclei in embryos from Ex oocytes at 96 h in the present study, for both glucose treatments (6.6–8.6), is similar to that which we have reported for Ex oocytes previously (6.5–7.7) (Choi et al. 2002a, 2003b). The average nucleus number of embryos from Cp oocytes, especially those from the high-glucose treatments (11.6 and 12.0) is notably higher than in our previous findings. This beneficial effect of glucose on embryos from Cp oocytes was not anticipated. Early embryos in other species have typically been shown to have better development in low glucose (Bavister 1995, Lane 2001), or no difference has been seen (Devreker et al. 2000). Further investigation on the effect of glucose during equine embryo development is warranted.

We were interested in evaluating the effect of temperature during manipulation for ICSI, as this may have an effect on the integrity of the meiotic spindle. Microtubules are extremely temperature-sensitive; in human oocytes, temperatures of even 3–4 °C less than 37 °C during manipulation significantly decreased the proportion of oocytes with identifiable spindles, and also fertilization and pregnancy rates (Wang et al. 2002). Low temperatures have been used during ICSI with the piezo drill to minimize oocyte lysis (Wakayama & Yanagimachi 1998a,b). However, in the present study, there was no difference in survival rate of oocytes subjected to ICSI at low and high temperature (97% and 95% respectively). While there was no apparent detrimental effect of low temperature on embryo development, if microtubule disorganization results in chromosomal anomalies, the effect may not be seen until further stages of development.

In conclusion, Ex oocytes had higher meiotic competence than did Cp oocytes at all durations of maturation tested. Normal cleavage rates were highest for Ex oocytes when matured for 24 h, but they were highest for Cp oocytes when matured for 30 h. Expanded oocytes matured for 24 h had a higher rate of normal cleavage after ICSI than did Cp oocytes at any duration of maturation; however, the embryo development of Cp oocytes 48 or 96 h after ICSI was significantly advanced over that for Ex oocytes. High glucose concentration in culture medium improved cleavage rates of Cp oocytes, but did not affect Ex oocytes. The piezo drill may be used for ICSI of equine oocytes at 37 °C without increasing oocyte lysis, thus allowing manipulation at a temperature that supports maintenance of the meiotic spindle.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This research was supported by the Link Equine Research Endowment Fund, Texas A&M University and Bio-Arts Research Corporation.

	Footnotes

 
Received 20 June 2003
First decision 28 August 2003
Revised manuscript received 1 October 2003
Accepted 27 October 2003

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