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BOR - Papers in Press, published online ahead of print January 8, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.009852
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BIOLOGY OF REPRODUCTION 68, 2065–2072 (2003)
DOI: 10.1095/biolreprod.102.009852
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Meiotic Competence of Equine Oocytes and Pronucleus Formation after Intracytoplasmic Sperm Injection (ICSI) as Related to Granulosa Cell Apoptosis1

Maria Elena Dell'Aquila2,3, Maria Albrizio3, Filippo Maritato3, Paolo Minoia3, and Katrin Hinrichs4

Department of Animal Production—Section of Reproductive Biology and Veterinary Obstetrics,3 University of Bari, 70010 Valenzano, Bari, Italy Department of Physiology and Pharmacology, College of Veterinary Medicine,4 Texas A&M University, College Station, Texas 77843-4466


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.

apoptosis, assisted reproductive technology, fertilization, gamete biology, granulosa cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proportion of horse oocytes maturing in vitro has varied among laboratories and is generally lower than those reported for other domestic species [1–14]. Typically, 40%–70% of equine oocytes reach metaphase II (MII) after culture for in vitro maturation, whereas in goats, sows, and cows, more than 90% of oocytes progress to MII (for a review, see Goudet et al. [15]). Much of the variation observed in vitro maturation (IVM) of horse oocytes may be explained by the selection of oocytes for culture. Horse oocytes originating from viable follicles have low meiotic competence [10] until the follicle reaches over 20 mm in diameter [12]. However, horse oocytes originating from follicles in early atresia have high meiotic competence (70%–86% maturation [10]). Cumulus expansion is strongly associated with follicle atresia in the horse [10, 16], and so selection for cumulus morphology can be used to approximate selection for atretic vs. viable follicles. Such selection is not completely accurate, however, as oocytes in early atresia may have either compact or expanded cumuli.

Oocytes with expanded cumuli (Exp oocytes; presumed to be from atretic follicles) not only mature in greater proportions than do oocytes with compact cumuli (Cp oocytes), they also mature more rapidly in culture [9, 17, 18] and activate more readily [19]. Because of the failure of in vitro fertilization (IVF) to be successfully repeatable in the horse [20], little work has been done on the developmental competence of different classes of horse oocytes. However, in two reports, the proportion of oocytes forming a male pronucleus after intracytoplasmic sperm injection (ICSI) was significantly higher in Exp than in Cp oocytes [5], and the proportion of normally fertilized oocytes after IVF was higher in Exp than in Cp oocytes [21]. When Exp oocytes were fertilized by ICSI and transferred to the oviducts of inseminated mares, 85% cleaved and embryos had developed to an average of 16 cells at 96 h after transfer, equivalent to normal development in vivo [22].

Molecular processes that result in oocyte meiotic progression from MI to MII are accompanied by a substantial increase in the activity of several kinases. The central components of this activity are the maturation promoting factor (MPF), a serine-threonine protein kinase composed of a regulatory subunit, cyclin B, and a catalytic subunit, p34cdc2, and the mitogen-activated protein kinase (MAPK). In all species studied, MPF activity appears shortly before germinal vesicle breakdown (GVBD), reaches a peak in MI oocytes, decreases dramatically during the transition from MI to MII, and regains its maximal level in MII oocytes. This pattern of changes implies that the transition from MI to MII is correlated with a decrease in MPF activity. MAPK is activated around GVBD, and its activity remains at high levels during the MI/MII transition. These two kinases are required for initiation and completion of meiosis and specifically for the regulation of microtubule dynamics leading to organization of the meiotic spindle, extrusion of the first polar body, and meiotic arrest at the MII stage (for reviews, see Day et al. [23], Anas et al. [24], Dell'Aquila et al. [25]).

The viable follicle keeps the oocyte in meiotic arrest, and release from the follicle and culture in vitro result in spontaneous resumption of meiosis in the majority of oocytes [26]. The follicular factor that maintains meiotic arrest is not known. In the mouse, hypoxanthine has been ascribed this role [27], but it does not appear to be applicable across species (for a review, see Sirard and Bilodeau [28]). The follicular suppressive factor is thought to work by inhibiting the activation of MPF. During follicle atresia, the meiotic suppressive factor appears to be lost, and oocytes may progress to MI and MII within severely atretic follicles [10, 26, 29, 30]. In the horse, progression to MII is seen only in follicles in end-stage atresia, in which the follicle is lined solely by a hyaline membrane. In such follicles, 22% of oocytes had resumed meiosis, 33% were degenerating, and 45% were still in the germinal vesicle stage [10]. In follicles in early to midstage atresia, 82% of oocytes were in the germinal vesicle stage; thus, over all atretic follicles, 76% of oocytes were in the germinal vesicle stage [10]. Follicles in early to midstage atresia appear to allow acquisition of meiotic competence of the enclosed oocyte, while oocytes within similarly sized viable follicles remain meiotically incompetent [12]. As discussed above, meiotic competence seen in equine Exp oocytes is accompanied by at least early developmental competence [22]. However, the relationship of developmental competence to the extent of follicle atresia is unknown.

Data from studies in different species suggest that granulosa cell (GC) apoptosis is the molecular mechanism underlying follicular atresia in mammals. Apoptosis is regulated by an array of extracellular signals through endocrine, paracrine, autocrine, or juxtacrine mechanisms in a development-dependent manner [31, 32]. Apoptosis of GC has been observed in rats [33–35], hens [36], cattle [37], pigs [38], sheep [39], and humans [40]. Apoptosis is the programmed death of a cell in the absence of an inflammatory reaction. All nucleated cells will initiate apoptosis upon stimulation by the appropriate signals. One of the features of this phenomenon, at the molecular level, is the activation of an endogenous Ca2+/Mg2+-dependent endonuclease that results in the fragmentation of cellular DNA into oligosomal length fragments. After agarose gel electrophoresis and staining with ethidium bromide, these fragments give a characteristic DNA ladder. Therefore, this event provides a marker for identification of apoptotic cells and measurement of the extent of apoptosis. In addition, in apoptosis, the nuclear envelope is irreversibly destroyed via proteolytic breakdown of the nuclear lamina, resulting in the formation of vesicular membranous structures containing nuclear fragments, termed apoptotic bodies, which may also be used as a marker for apoptosis (for a review, see Terranova and Taylor [41]).

Few reports are available to date on apoptosis in horse follicles. Okolski et al. [42] evaluated equine GCs for presence of nuclear fragments they termed atretic bodies; presence of these bodies was correlated with reduced estrogen levels in the follicular fluid but was not correlated with the proportion of oocytes that matured in vitro. Pedersen et al. [43], using DNA laddering, found that small follicles were more likely than were large follicles to be apoptotic, to have Exp cumuli, and to have condensed chromatin within the germinal vesicle of the enclosed oocyte. Albrizio et al. [44] found that small follicles from anestrous mares had high a incidence of apoptosis (80% apoptotic bodies [AB] and a high incidence of DNA laddering) whereas those from transitional, estrous, and diestrous mares showed a lower incidence of apoptosis (30%–40% AB and an intermediate incidence of DNA fragmentation). Large preovulatory follicles did not show DNA fragmentation or AB. Sciorsci et al. [45] found that treatment with opioid antagonists reduced the prevalence of apoptotic follicles in anestrous mares. No information is available on the relationship of GC apoptosis to maturation and early developmental competence in horse oocytes. Recent studies in cattle and sheep indicate that oocyte competence decreases only at a high level of follicular atresia, while it appears to be improved by a low level of atresia [46–50].

The objective of our study was to evaluate the degree of apoptosis in different categories of horse follicles by DNA laddering analysis and to determine the relationship of GC apoptosis to meiotic competence and cytoplasmic maturation (ability to achieve normal pronucleus formation after ICSI) of horse oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

This study was conducted in southern Italy (41°N) during the breeding season (April–October). Oocytes were recovered individually from small (5–9 mm in diameter), medium (10–25 mm), and large (>25 mm) horse follicles. Granulosa cells from the follicle were processed to extract DNA, which was evaluated for detection of laddering. The recovered oocyte was evaluated for cumulus morphology and cultured for IVM. Those oocytes having a first polar body (PB) after culture were subjected to ICSI and cultured for 24 h after sperm injection to evaluate pronucleus formation.

Collection of Oocytes and Granulosa Cells

Ovaries from mares of unknown reproductive history were obtained at two local abattoirs located at a maximum distance of 20 km (30 min) from the laboratory. The ovaries were placed in physiological saline (9 g NaCl containing 40 mg/L gentamycin sulphate) within 30 min of slaughter and were transported to the laboratory in thermal containers set at 30°C (1–2 h transport). All antral follicles that were visible on the ovarian surface were measured with a ruler, then opened with a scalpel blade and the GC layer scraped with a curette following the method of Hinrichs et al. [9]. Granulosa cells were flushed from the curette into individual Petri dishes using HEPES-buffered tissue culture medium (medium 199, Gibco BRL; Life Technologies Ltd., Paisley, Scotland) supplemented with 10% fetal calf serum (F-4135; Sigma, Milan, Italy). Cumulus-oocyte complexes (COCs) were identified in the collected mural GC using a dissection microscope. Cumulus-oocyte complexes, classified as having compact (Cp), expanded (Exp), or partial (P) cumulus investment, were selected for culture; degenerating oocytes (having shrunken, dense, or fragmented cytoplasm) were recorded and discarded from further processing. Those oocytes selected for culture were washed four times in the same medium. The time between follicle scraping and starting of oocyte culture or GC freezing was less than 1 h, and the total time between slaughter and culture ranged between 2 and 4 h.

In Vitro Maturation

In vitro maturation was performed using medium TCM-199 (M-2154; Sigma) with Earle salts buffered with 4.43 mM HEPES (H-9136; Sigma) and 33.9 mM sodium bicarbonate (S-5761; Sigma) and supplemented with 0.1 g/L l-glutamine (G-7513; Sigma), 2 mM sodium pyruvate (P-2256; Sigma), 2.92 mM calcium lactate (No. 29760; Serva Feinbiochem GmbH & Co., Heidelberg, Germany), and 50 µg/ml gentamycin (G-1272; Sigma). After preparation, pH was adjusted to 7.18, and the medium was filtered through 0.22-µm filters (No. 5003-6; Lida Manufacturing Corp., Kenosha, WI). Then gonadotropins (10 µg/ml ovine FSH [Sigma] and 20 µg/ml ovine LH [NIH, Bethesda, MD]) and 17ß-estradiol (1 µg/ml, E-2257; Sigma) were added. The medium was further supplemented with 20% (v/v) fetal calf serum (F-4135; Sigma). The medium was filtered again and allowed to equilibrate for 1 h under 5% CO2 in air before being used. Each single COC was placed in a numbered 25-µl microdrop of medium in a 30-mm Petri dish, covered with preequilibrated lightweight paraffin oil (M-3516; Sigma), and cultured for 28–30 h at 38.5°C under 5% CO2 in air.

Oocyte Evaluation and Preparation for ICSI

After 24–26 h IVM culture, oocytes were denuded of cumulus and corona cells by incubation in TCM199 with 20% FCS containing 80 IU hyaluronidase/ml (H-3506; Sigma) and by aspiration in and out of finely drawn glass pipettes. Oocyte morphology was assessed and those oocytes showing the perivitelline space, an extruded first PB, and an intact oolemma were selected and submitted to microinjection [5], whereas those oocytes not showing the first PB were fixed and stained to evaluate the stage of nuclear maturation.

Semen Preparation for ICSI

Semen samples (0.4 ml/straw) from a single ejaculate frozen at a concentration of 1 x 108 sperm cells/ml were rapidly thawed (30 sec) in a water bath at 37°C. Total motility after thawing was 70%, with 50%–60% progressive motility. Sperm cells for ICSI were prepared by the swim-up procedure in Earle balanced salt solution (E-2888; Sigma) supplemented with 0.4% BSA, 40 µg/ml streptomycin sulphate (S-9139; Sigma), and 25 UI/ml penicillin G (P-3032; Sigma) as described by Dell'Aquila et al. [5].

ICSI Procedure

Intracytoplasmic sperm injection was carried out according to the procedures described by Palermo et al. [51] and Van Steirteghem et al. [52] as applied to equine germ cells by Dell'Aquila et al. [5]. All procedures were performed at 38.5°C in HEPES-buffered human tubal fluid (HTF-HEPES 9962; Irvine Scientific, Santa Ana, CA). The injected oocytes were then transferred to 25-µl drops of fresh HTF medium covered by lightweight paraffin oil and incubated at 38.5°C for 18–20 h under 5% CO2 in air.

Nuclear Chromatin Evaluation of Zygotes

On the day after microinjection, oocytes were assessed for signs of fertilization. To evaluate nuclear chromatin, oocytes were fixed in 3.8% buffered formaldehyde solution (no. 7385; J.T. Baker), stained with 2.5 µg/ml Hoechst 33258 (B-1155; Sigma) in 3:1 (v/v) glycerol/PBS, and observed under an E-600 Nikon fluorescent microscope with a 365-nm excitation filter. Normal fertilization was defined by the presence of two PBs with two pronuclei (PN; Fig. 1). Oocytes found cleaved (two- to four-cell stage), showing two to four regular-shape blastomeres with a normal nucleus for each blastomere, were also considered to be normally fertilized (Fig. 2). Presence of a swollen sperm head, a single PN with signs of the sperm cell in the cytoplasm, tripronucleate zygotes (3PN) with a single polar body extruded and abnormal cleavage (irregular shape blastomeres with irregular number of nuclei) were considered to represent retarded, arrested, or abnormal fertilization and were grouped together as abnormal. Parthenogenetically activated oocytes showing one PB and one PN and oocytes showing the metaphase II with or without the intact sperm cell were classified as unfertilized.



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  		FIG. 1. Equine oocyte recovered from a follicle showing an intermediate level of apoptosis and observed after successful fertilization by ICSI. Two pronuclei (PN) in the ooplasm and two polar bodies (PB) in the perivitelline space are visible (Hoechst 33258 stain). Bar = 50 µm



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  		FIG. 2. Two- to four-cell stage equine embryo produced by ICSI, derived from a follicle in intermediate level of apoptosis and showing two sets of mitotic chromosomes observed at the end of the telophase (arrows). Two polar bodies (PB) in the perivitelline space are also visible (Hoechst 33258 stain). Bar = 50 µm

Nuclear chromatin status of oocytes excluded from the ICSI procedure was classified as follows in relation to the stage at which meiotic development was interrupted: germinal vesicle (GV), metaphase to telophase I (MI), and metaphase II (MII) with or without the chromatin of the first PB. Oocytes showing either multipolar meiotic spindle or irregular chromatin clumps or no chromatin were considered to be abnormal. Oocytes with fragmented or shrunken cytoplasm were classified as degenerated.

Identification of Apoptosis

Granulosa cells from each follicle were collected from the Petri dish containing the follicular contents. The recovered tissue was centrifuged at 400 x g for 10 min and the supernatant was discarded. The resulting pellet was weighed, frozen in liquid nitrogen, and stored at -80°C until molecular analysis.

DNA Extraction

Cells were thawed and resuspended in 200 µl phosphate buffered saline (Sigma P-4417) and the DNA was extracted using the Apoptotic DNA Ladder Kit (Roche Diagnostics S.p.A., Milan, Italy) following the manufacturer's instructions. The amount of DNA extracted was quantitated by spectrophotometric absorbance at 260 nm.

Detection of Apoptosis by DNA Laddering

Five micrograms of DNA were loaded per lane on 2% agarose gels and electrophoretically separated in 1x TAE (40 mM Tris-acetate, 1mM EDTA) at 80 V for 1.5 h. The DNA extracted from the apoptotic U937 cell line was used as the apoptotic marker. After DNA migration, gels were stained with ethidium bromide (1 µg/ml) and the DNA fluorescence was viewed with an ultraviolet transilluminator. The intensity of band fluorescence was measured using the Gel Doc 2000 documentation system (Bio-Rad Laboratories S.r.l., Milan, Italy), and data were analyzed by the Quantity One (Bio-Rad) software. For each sample, the incidence of DNA fragmentation was evaluated by comparing the concentration of three selected bands, chosen in the analyzed ladder patterns, with the concentration curve obtained from the apoptotic U937 marker. The three bands were selected to follow the degree of chromatin fragmentation by measuring the intensity of the band corresponding to the undigested DNA (>15 kbp) and the intensity of a band of low (360 bp) and intermediate molecular weight (900 bp), generated by the activity of endonucleases.

A sample was considered as

  1. apoptotic when both the 900- and the 360-bp bands were present and the concentration of the 360-bp band was higher than that of the 900-bp band;
  2. intermediate when both the 900- and the 360-bp bands were present and the concentration of the 900-bp band was higher than that of the 360-bp band;
  3. nonapoptotic when the intensities of the 900- and the 360-bp bands were either not detectable or lower that 5 units compared with the intensity of the undigested DNA.

Analysis of Data

The proportion of oocytes in MII after maturation and the proportion of fertilized oocytes after ICSI were compared between groups of oocytes having different cumulus morphologies and issuing from healthy, intermediate, or apoptotic follicles by chi-square analysis with the Yates correction for continuity. The Fisher exact test was used when a value of less than five was expected in any cell. Effects of follicle size on cumulus morphology and on granulosa cell apoptosis were analyzed by linear regression analysis. Differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oocyte Recovery

From the ovaries of 30 mares, 231 follicles were scraped (3.85 follicles/ovary) and 110 oocytes were recovered, for a recovery rate of 48%. A total of 98 follicle-oocyte pairs were examined. Twelve samples were excluded from the analysis in that eight of them had the oocytes lost or damaged during handling before evaluation and culture and four of them did not have enough GC cells for molecular analysis.

Follicle Size and Cumulus Morphology Evaluation

The 98 follicles ranged from 5 to 50 mm in diameter. The number of follicles in each category were small, n = 22; medium, n = 67; and large, n = 9. The numbers and percentages of oocytes showing Cp, Exp, or P cumulus for each size category are shown in Table 1. There were no significant effects of follicle size on cumulus morphology (R2 = 0.0258, P > 0.05). Overall, 39 follicles (40%) contained a Cp-cumulus oocyte, 52 (53%) had an oocyte surrounded by an Exp cumulus, and 7 (7%) contained an oocyte with a P cumulus.


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  		TABLE 1. Relationship between follicle size and cumulus morphology in the mare.*

Follicular Wall Apoptosis and Cumulus Morphology

Samples were classified according to the degree of chromatin fragmentation, defined as the presence of the complete DNA ladder pattern, and as the concentration, expressed as referred to the apoptotic DNA marker, of a 360- and a 900-bp fragment as described in the Materials and Methods. The three observed patterns of DNA fragmentation are shown in Figure 3. The numbers of follicles classified as showing advanced or intermediate apoptosis or as nonapoptotic are presented in Table 2. Significantly more oocytes with Exp than with Cp cumuli originated from follicles in the advanced stage of apoptosis (P < 0.001). Only one oocyte was degenerated at the time of retrieval, and it issued from an apoptotic medium-sized (20-mm) follicle and was surrounded by an Exp cumulus.



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  		FIG. 3. Representative gel of samples showing different levels of internucleosomal DNA fragmentation. Five micrograms of DNA, isolated from granulosa cells, were applied to each lane of a 2% agarose gel and electrophoresed as described in the Material and Methods. Arrows show fragments chosen for the densitometric analysis. Lane 1: DNA from a follicle showing an intermediate level of apoptosis; lane 2: DNA from an apoptotic follicle; lane 3: DNA from a healthy follicle; lane M: marker DNA from the apoptotic U 937 cell line


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  		TABLE 2. Relationship between granulosa cell apoptosis and cumulus morphology in the mare

Follicular Wall Apoptosis as Related to Follicle Size

The relation between follicle size and apoptosis is shown in Figure 4. There was no significant effect of follicle size on the degree of granulosa cell apoptosis (R2 = 0.0128, P > 0.05).



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  		FIG. 4. Graphic representation of the correlation between apoptosis of mural granulosa cells and follicle size in the mare. Columns represent the three follicle size categories: small, medium, and large. The number of follicles found in each size category was small, n = 22; medium, n = 67; and large, n = 9. Within each column, the proportion of oocytes issuing from apoptotic, intermediate, or healthy follicles is indicated. Numbers within each bar area indicate the number of follicles found to belong to each size category and level of apoptosis. Linear regression analysis indicated no significant effects of follicle size on the levels of apoptosis (R2 = 0.0128, P > 0.05)

In Vitro Maturation of Equine Oocytes as Related to Follicular Wall Apoptosis

Nuclear maturation after culture for IVM was determined by extrusion of the first polar body because these oocytes were subjected to ICSI without fixing and staining. The proportion of oocytes that matured in culture in relation to their initial cumulus morphology and degree of GC apoptosis is presented in Table 2. A significantly higher proportion of oocytes from follicles in advanced apoptosis had reached MII after culture than was observed in oocytes from nonapoptotic follicles (39/54 [72%] vs. 2/8 [25%]; P < 0.05).

Pronuclear Formation after ICSI as Related to Granulosa Cell Apoptosis

The proportion of normally fertilized oocytes after ICSI did not differ significantly in relation to GC apoptosis, nor did it differ among cumulus types (Table 3). Only one of two MII oocytes from nonapoptotic follicles was fertilized after ICSI; this oocyte had an expanded cumulus at the time of retrieval. The proportion of abnormally fertilized oocytes was significantly higher in P than in Cp oocytes in the apoptotic group (P < 0.05; Table 4).


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  		TABLE 3. Proportions of matured and normally fertilized equine oocytes after ICSI in relation to cumulus morphology and granulosa cell apoptosis.*


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  		TABLE 4. Proportion of abnormally fertilized and unfertilized equine oocytes after ICSI in relation to cumulus morphology and granulosa cell apoptosis.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first in the horse to show that granulosa cell apoptosis is related to the meiotic competence of horse oocytes. The significant relationship between cumulus expansion and GC apoptosis is expected, as cumulus expansion is associated with follicle atresia [10]. Similarly, the increased capacity for maturation in vitro of oocytes from apoptotic follicles reflects that previously observed in oocytes from follicles in early and midstage atresia [10].

We hypothesized that the high level of advanced and intermediate apoptosis that we found in this study may be related to the variable time intervals between death and GC collection or freezing for DNA analysis (2–4 h). We performed correlation/regression analysis (98 observations) to evaluate the degree of apoptosis as a function of the time from slaughter, arrival to the laboratory, GC and oocyte recovery, placement of the oocytes into culture, and freezing of related GCs. Simple linear regression analysis indicated that the time from slaughter to final preservation of GCs did not significantly influence the apoptosis level (R2 = 0.0035, P > 0.05). These data were confirmed by intraclass evaluations for Cp, Exp, and P oocytes, respectively. This suggests that use of slaughterhouse tissue after a 2–4-h delay is acceptable for evaluation of follicle apoptosis. Use of slaughterhouse material, with over 2 h elapsing between slaughter and analysis, has been standard in studies of follicular apoptosis in other species [48, 49, 53]. Moreover, Boone et al. [54] reported that, in rats, DNA laddering in GCs was not increased for up to 24 h after ovary excision.

In the present study, there was no difference in the maturation rate among oocytes with differing cumulus morphology. While we did not assess the germinal vesicle chromatin of oocytes, previous studies have reported that only a small percentage of Exp oocytes are in MI or MII at the time of recovery (9%–12%, Hinrichs and Schmidt [12]; 7%, Hinrichs et al. [9]; 11%, Hinrichs and Williams [10]). There is some disagreement among laboratories on the relationship of cumulus morphology to meiotic competence in horse oocytes: in some reports, similar proportions of Cp and Exp oocytes were found to mature in vitro [5], whereas in others, significantly higher proportions of Exp oocytes than of Cp oocytes matured [9]. Cumulus morphology is assessed subjectively under low magnification and thus is subject to variation among operators; oocytes represented in the compact and expanded cumulus groups may therefore differ among laboratories. The significant relationship between meiotic competence of horse oocytes and more objective measures, such as atresia as evaluated on histological section [10] and apoptosis as evaluated by DNA laddering (this study), are strong indicators that meiotic competence in the horse is acquired during follicle atresia.

The technique of DNA laddering was employed because internucleosomal DNA fragmentation, detected by agarose gel electrophoresis, is considered to be a very specific marker of apoptosis and an event that occurs early in the cell death process, preceding morphological changes leading to the formation of apoptotic bodies [55, 56]. The amount of degraded DNA, especially within the range of mono and oligonucleosomal DNA fragments, increases with progression of apoptosis [57].

No references are currently available on densitometric analysis of DNA fragments in ladder patterns of granulosa cells in the horse. Therefore, we carried out the analysis by applying criteria reported for other species [49, 54, 58] or other tissues [59] based on the fact that cells undergoing apoptosis cleave their DNA between nucleosomes, yielding fragments in multiples of 180–200 bp. Moreover, because a moderate apoptotic level was previously reported in bovine follicular wall [49], we followed the relative concentration of an intermediate molecular weight band (900 bp) in respect to a low molecular weight band (360 bp).

The association between GC apoptosis and meiotic competence observed in this study can be related to previous observations in other species. In the sheep, a positive effect of early atresia on oocyte competence has been found for follicles of 1–3 mm [47]. In the cow, it has been reported that mild follicular atresia does not negatively affect oocyte competence [46] and that follicles with more than 73% of the cells being apoptotic can still yield a competent oocyte [48]. However, Jewgenow et al. [49] reported that reduced oocyte developmental capacity is related to apoptotic death of follicular cells even before morphological signs of severe atresia are detectable. These authors also reported that single oocyte maturation reduced developmental capacity of oocytes issuing from apoptotic follicles compared with group culture. This was not the case in our study, in which the proportion of oocytes maturing in vitro and forming pronuclei after ICSI (72% and 44%, respectively) when cultured individually were comparable with those of previous studies (for a review, see Dell'Aquila et al. [7]).

Within oocytes that reached MII, there was no relationship between apoptosis and cytoplasmic maturation, i.e., the ability of the oocyte to activate normally after fertilization. The evaluation of the relationship between apoptosis and normal fertilization was hindered in this study by the small number of oocytes in the nonapoptotic group and their low rate of maturation (2/8, 25%). Thus, a meaningful comparison of the proportion of fertilized oocytes from nonapoptotic follicles as compared with those from intermediate and apoptotic follicles was not possible. In addition, data on oocytes from the most atretic follicles (stage 3, Kenney et al. [60]) may not have been included, as these follicles are lined by only a hyaline membrane and may not have provided enough GCs to allow DNA analysis.

These data represent the first findings on the relationship between follicle status and early developmental competence in the horse. Because equine IVF has failed to be successful, it was not possible to evaluate developmental competence in horse oocytes until repeatable ICSI procedures were developed. Lack of effective methods for in vitro culture of equine embryos still limits our ability to determine the extent of developmental competence of equine oocytes. For example, it is possible that, while similar proportions of oocytes from intermediate and apoptotic follicles activated after ICSI in the current study, further competence (i.e., for cleavage and embryo development) may have been different among groups. Investigation of embryo development in different classes of equine oocytes should be performed once culture systems have been optimized in this species. The results of this and future studies may aid in the appropriate selection of horse oocytes for assisted reproductive techniques, similar to that presently done in cattle, so that blastocyst production and maintenance of pregnancy after transfer of IVP embryos may be maximized.

The impact of follicular apoptosis on the outcome of assisted reproductive programs has been widely studied in human reproductive medicine. In one study, follicular wall apoptosis was not related to fertilization or embryo development [61]. In contrast, another study reported that a high incidence of apoptotic bodies in the membrana granulosa was associated with poor IVF outcome and that apoptotic bodies can be used as a predictive marker in assisted fertilization programs [40]. It should be noted that these programs utilize in vivo-matured oocytes collected from preovulatory follicles in gonadotropin-stimulated women; thus, direct comparison to data from immature follicles is not possible [61]. In human oocytes surrounded by cumulus cells showing high incidence of apoptosis, rates of fertilization and embryo development are higher with ICSI than with conventional IVF [62]. This is attributed to the higher concentration of sperm enzymes associated with conventional IVF, which contributes to harsh culture conditions. More meaningful comparisons with results in humans could be derived from data on ex vivo collected, mature oocytes in the mare.

Apoptosis of cumulus cells surrounding the oocyte, rather than of mural GCs, may be performed using techniques to handle a small number of cells and detect early stages of apoptosis [63–65]. The cumulus-oocyte complex is the last part of the follicle that is affected by follicular atresia [66, 67]. The relationship between cumulus cell apoptosis, mural GC apoptosis, and oocyte developmental competence could be of particular importance in the equine species, which is characterized by peculiarly large follicles. It has been reported that bovine oocytes with mildly atretic cumulus cells yield a higher blastocyst rate than oocytes without visibly atretic signs [46, 67] and that developmental competence is only affected by advanced atresia of the cumulus-oocyte complex [46, 67]. In humans, it has been reported that apoptosis in cumulus cells is significantly correlated to maturation stage, fertilization rate, and embryo score of the corresponding oocyte [65, 68] and that the incidence of cumulus cell apoptosis can be used in predicting oocyte quality, outcome of IVF-ET, and age-related decline in fertility [69].

In conclusion, this study demonstrates the presence of a high incidence of apoptosis in ovarian follicles in the mare that does not correlate with follicle size. Follicular apoptosis was related to cumulus expansion and increased meiotic competence but did not influence cytoplasmic maturation, as assessed by the proportion of oocytes that formed pronuclei after ICSI. Further investigation is needed to provide information on the effects of GC apoptosis on subsequent embryonic and fetal development in the mare.


   ACKNOWLEDGMENTS
 
The authors thank Prof. Alessandro Leopold and Prof. Gaetano Mari (Institute of Artificial Insemination, Cadriano, University of Bologna, Italy) for giving us frozen stallion semen and the NIADDK, NIH (Bethesda, MD) for providing ovine LH.


   FOOTNOTES
 
1 Work by K.H. was supported by the Link Equine Research Endowment, Texas A&M University; work by P.M. and coworkers was supported by CEGBA, University of Bari, Italy. Part of this work was presented at the Annual Conference of the International Embryo Transfer Society, Maastricht, The Netherlands, 9–11 January 2000, and at the LIV Meeting Società Italiana Scienze Veterinarie, Riva del Garda (TN), Italy, 28–30 September 2000. Back

2 Correspondence: Maria Elena Dell'Aquila, Department of Animal Production—Section of Reproduction, Faculty of Biotechnological Sciences, University of Bari, Str. Prov. Casamassima Km 3, 70010 Valenzano, Bari, Italy. FAX: 39 080 4679883; e.dellaquila{at}veterinaria.uniba.it Back

Received: 10 October 2002.

First decision: 28 October 2002.

Accepted: 6 January 2003.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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