Meiosis Resumption of Canine Oocytes Cultured in the Isolated Oviduct

Introduction

The canine oviduct supports long term survival of oocytes that are ovulated in the dictyate stage and that in this tract complete maturation, undergo fertilization and develop up to the blastocyst stage (Holst and Phemister 1971). Extra-follicular maturation and extended time of survival in the oviduct are peculiarities of Canid oocytes.

It is well-known that a successful in vitro system for oocyte maturation should be based on in vivo environmental conditions, thus, in the domestic dog, the attention should be focused on the oviductal features. However, precise knowledge on the canine oviductal environment has not been acquired yet and, consequently, the definition of an in vitro system for culturing canine oocytes is still far from being achieved.

In the last few years, research has been directed towards the identification of adequate cultural conditions for canine oocytes (Farstad 2000; Luvoni 2000). Different media with or without serum or hormonal supplementation have been used (Hewitt and England 1997, 1999a; Hewitt et al. 1998; Otoi et al. 1999; Songsasen et al. 2002; Rodrigues and Rodrigues 2003a), but none of them allowed the achievement of the in vitro maturation efficiency reached in most other domestic animals. Even the culture time required for the completion of nuclear maturation is still under investigation, some authors showed that in 24–48 h full nuclear maturation occurs (Nickson et al. 1993; Bolamba et al. 1998; Saint-Dizier et al. 2001) while others adopted a longer time of culture up to 72–96 h (Mahi and Yanagimachi 1976; Yamada et al. 1992; Hewitt et al. 1998; Fujii et al. 2000; Otoi et al. 2000). However, the extension of the culture time increases oocyte degeneration and the efficiency of in vitro maturation remains very low. Consequently, limited success in terms of oocyte fertilization and embryo development in vitro has been achieved in the dog, and only one pregnancy has been reported after transfer of in vitro fertilized oocytes (England et al. 2001).

Attention on the oviductal environment was first focused on by Hewitt and England (1999b). They reported an improvement of meiosis resumption rates of canine oocytes cultured either in synthetic oviduct fluid (SOF, Tervit et al. 1972) supplemented with a high concentration of proteins, or in presence of oviductal cells, but only after a prolonged culture time (96 h). Subsequently, Bolamba et al. (2002) demonstrated that culture for 48 and 72 h in the simple medium SOF, without the addition of any protein source, supported in vitro nuclear maturation of bitch oocytes, although at low rates. Moreover, Bogliolo et al. (2002) evaluated the effect of co-culture with canine infundibulum and ampulla oviductal cells collected during the oestrous cycle. They found a positive influence of the cells on the meiotic resumption and full nuclear maturation of canine oocytes after 48 and 72 h of culture.

Oviductal tissue has been successfully used as a co-culture for a variety of mammalian oocytes as it is known that the physical interaction between the oocytes and the oviductal epithelium is important in vivo (Elliott 1974) and it enhances developmental competence of the oocytes in vitro (Gandolfi and Moor 1987).

The co-culture of canine oocytes on a monolayer of oviductal cells is an attempt to create the physiological conditions in which they resume meiotic maturation, however this model is still far from the in vivo oviductal environment that, in the dog, has some unique qualities that are beneficial for supporting maturation and extending viability of the oocytes.

Therefore, the aim of this study was to investigate the benefits of the culture of canine oocytes in isolated oviducts in the attempt to mimic the in vivo physical interaction and spatial arrangement between the oocytes and the oviductal tract. The effects on meiotic maturation, on the time required to resume meiosis and on the viability of the oocytes were evaluated.

Materials and Methods

Chemicals

All chemicals in this study were purchased from the Sigma Chemical Company (St Louis, MO, USA) unless otherwise stated.

Collection of canine oocytes and oviducts

Ovaries within the ovarian bursa were harvested from pubertal bitches at random stages of the oestrous cycle during routine ovariohysterectomy at local veterinary clinics. The bitches of various breeds and aged 1–8 years were all healthy at the time of surgery. Ovaries within the ovarian bursa were stored at 38°C in phosphate-buffered saline (PBS) with a mixture of antibiotics and antimycotics (AB: 100.000 i.u. penicillin G sodium/ml, 100 mg streptomycin sulphate/ml; AM: 250 μg amphotericin B/ml) for 1–2 h before processing. Ovaries were sliced in PBS and AB with 0.1% (w/v) polyvinyl alcohol to release cumulus–oocyte complexes (COCs). Only COCs with darkly pigmented ooplasm, completely surrounded by one or more layers of cumulus cells (grade 1), were selected for the experiments and rinsed in maturation medium before culture. Oviducts were dissected free from connective tissue and isthmus–ampullar tracts were isolated, rinsed in PBS with AB/AM, and prepared in two different ways for culture of canine oocytes, before being placed in a Petri dish with maturation medium. Some oviductal tracts were prepared by making a longitudinal incision with a surgical blade in order to expose the mucosal epithelium (open oviduct) on which canine oocytes were cultured. Some others were left intact and a ligature was placed at one end with surgical thread (ligated oviduct). Cumulus–oocyte complexes were loaded into a glass pipette with a minimal amount of maturation medium and the open end of the oviduct was catheterized to deposit the COCs, before placing a second ligature on it.

After culture, the oocytes were recovered with a glass pipette from the open oviduct and by cutting one ligature and squeezing the oviduct from the ligated one.

In vitro maturation

The maturation medium was Tissue Culture Medium 199 (TCM 199; M4530) with AB, 5% of foetal bovine serum, 0.5 iu FSH and 0.5 iu LH/ml (Pluset, Serono, Rome). Cumulus-oocyte complexes and oviducts were cultured in Petri dishes and incubated at 38.5°C, 5% CO₂ in air.

In experiment 1, the oviducts of each bitch were divided into two parts and used as an open or ligated oviduct, the pool of the COCs obtained from each pair of ovaries was divided into six groups to be respectively cultured for 24 and 30 h: (1) in 100 μl drops under oil (up to ten oocytes per drop); (2) on the mucosal epithelium of the open oviduct (up to 10 oocytes per oviduct); (3) in the ligated oviduct (up to 20 oocytes per oviduct).

In experiment 2, each oviduct of a bitch was cut in two parts and three of the four parts were used as a ligated oviduct, the pool of the COCs obtained from each pair of ovaries was divided into four groups to be respectively cultured in the ligated oviduct for 24, 30 and 48 h (up to twenty oocytes per oviduct). A group of control COCs was not cultured (0 h).

Assessment of oocyte nuclear maturation

Assessment of nuclear maturation was carried out after each incubation interval.

The procedure involved the removal of cumulus cells by vigorous shaking of the oocytes in 1% sodium citrate solution or by mechanical displacement with a small-bore glass pipette depending on cumulus expansion degree and two different staining procedures were used.

In experiment 1, denuded oocytes were fixed overnight in a freshly prepared 1 : 3 acetic acid : ethanol solution before being stained with 1% aceto-orcein to evaluate under a light microscope at 400× magnification the nuclear stage of maturation. According to the description reported by Bolamba et al. (2002) oocytes were classified as intact germinal vesicle (GV), GV breakdown (GVBD), diakinesis (DK), metaphase I (MI) through metaphase II (MII).

In experiment 2, oocytes were staining with bis-benzimide (Hoechst 33342), a DNA-specific fluorochrome, that allows a better evaluation of the chromatin configurations of oocytes after culture. Briefly, denuded oocytes were stained with a solution of sodium citrate : ethanol (3 : 1) of bis-benzimide (10 μg/ml). Oocytes were placed on a slide with a minimum amount of medium, then 10 μl of Hoechst solution were placed on them. After 5 min of incubation in the dark, the Hoechst solution was removed and the oocytes were covered with an antifade reagent (FluoroGuard, Bio-Rad, Hercules, CA, USA). The oocytes were overlaid with a coverslip supported by four droplets of vaseline/paraffin and were observed under a fluorescent microscope (Axiovert 100, Zeiss, Italy) at 400× magnification for chromatin configuration evaluation.

The oocytes were classified as follows: GV when nucleolus and very fine filaments of chromatin were identified; GVBD–DK when different patterns of chromatin condensation were present or when the chromosomes appeared coiled up and no individual chromosomes were visible; MI–MII once the formation of bivalents was completed and appeared one or two sets of chromosomes.

With both staining procedures, nuclear stages ranging from GVBD and MII were considered to have resumed meiosis and oocytes with nuclear material not classifiable or faintly stained or invisible, were considered as degenerated.

Assessment of oviductal morphology

In order to evaluate the morphology of the oviductal epithelium during culture, at the time of the recovery and after 24, 30 and 48 h of culture, oviducts were fixed and stained for microscopical evaluation. Briefly, oviducts were cut into small pieces (1–2 mm), fixed by immersion for 2 h at 4°C in 2.5% (v/v) glutaraldehyde in PBS (pH 7.4), dehydrated in ethanol and embedded in Epoxy resin (Epon 812, Polysciences, Inc. Warrington, PA, USA). Serial sections (1 μm in thickness) were stained with toloudine blue and examined with a conventional light microscope.

Statistical analysis

The percentages of different stages of nuclear maturation and degeneration of oocytes between treatments at each culture period, and between periods of culture within the same treatment, were analyzed using chi-square test. A confidence level of p < 0.05 was considered statistically significant.

Results

A total of 990 grade 1 COCs were obtained from 17 bitches (29.1 ± 14.3 SD oocytes per ovary) and 659 oocytes were selected for this study. The nuclear morphology was evaluated in 564 oocytes and the remaining oocytes were either broken during removal of cumulus cells and preparation of staining, or lost and not recovered from the ligated oviducts following culture.

Experiment 1

The results of the oocyte in vitro maturation in drop, open and ligated oviducts for 24 and 30 h of culture are summarized in Table 1.

At 24 h of maturation, proportions of oocytes that remained at the GV stage were significantly higher (p < 0.05) in drop and in the open oviduct than in the ligated oviduct, while at 30 h no differences were observed between the treatment groups. At 24 h and 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct than in drop or in the open oviduct, without differences between the two time intervals. However, at 24 h of culture, all the oocytes that resumed meiosis in the ligated oviducts were at GVBD–DK stage, but after 30 h of culture, 31.9% of the oocytes showed meiotic progression with nuclear stages ranging between MI and MII. This proportion was significantly higher (p < 0.05) than that obtained in the groups cultured in drop or in the open oviduct. Moreover, oocytes recovered from the ligated oviduct showed a better cumulus expansion than those cultured in the other systems.

At 24 and 30 h of maturation, oocyte degeneration was significantly lower in the ligated oviduct than in drop (p < 0.05) or in the open oviduct (p < 0.01).

Experiment 2

The results of the oocyte in vitro maturation in the ligated oviducts for 0, 24, 30 and 48 h of culture are summarized in Table 2.

High proportions of oocytes were at the GV stage at the time of collection (0 h) and a significant decrease was observed after 24, 30 and 48 h of culture (p < 0.0001).

Oocytes cultured in the ligated oviducts resumed meiosis at higher rates after 24 and 30 h (p < 0.005) than after 48 h of in vitro maturation. Resumption of meiosis was mainly determined by oocytes at the GVBD–DK stage rather than by oocytes at MI–MII stages. In fact, oocytes that reached the MI–MII stage, although in higher proportions at 24 and 30 h than at 48 h (p < 0.005), ranged between 12 and 14%.

Degeneration of the oocytes at 24 h of culture was similar to the uncultured oocytes, but it increased significantly (p < 0.0001) with the extension of the period of culture, reaching 59.3% at 48 h.

Oviductal morphology

At the time of collection (0 h) mucosal folds were lined by a simple columnar epithelium characterized by well-differentiated ciliated and secretory cells. Secretory cells showed a basally located nucleus and apical cytoplasmic protrusions, often extended beyond the ciliated cells. Hypertrophic cells with a large nucleus were found between columnar cells. Following culture, secretory cells were preserved at the bottom of the mucosal folds, while on the top, evident signals of dedifferentiation and degeneration of the epithelial cells were found.

Discussion

The present study suggests that the physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct. The ligated oviduct, however, does not assure viability of the oocytes up to 48 h of culture.

The oviductal environment in vivo is the site where ovulated canine oocytes survive longer than other mammalian oocytes, because in this tract oocytes undergo the completion of nuclear maturation other than the fertilization (Holst and Phemister 1971). This fact suggests that the bitch oocytes should be matured in vitro in the presence of oviduct cells or in a culture medium similar to the oviduct fluid as SOF. Culture conditions possessing these characteristics have been used by some authors with conflicting results. Hewitt and England (1999b) reported that SOF or oviductal cells do not exert any beneficial effect on the achievement of full nuclear maturation of bitch oocytes. In fact, they recorded a low percentage (9%) of oocytes maturing from MI–MII and this rate was not different from that observed in the culture without cells. An improvement on resumption of meiosis was observed, but only after 96 h of co-culture. On the other hand, Bogliolo et al. (2002) demonstrated that co-culture in TCM 199 with oviductal cells collected from ampullar tracts of bitches in oestrous, had a beneficial effect on the oocyte maturation rates (MII stages) that reached 16.7 and 23.2%. Oocytes at meiotic stages ranging between MI and MII were 32.5 and 29% after 48 and 72 h of culture.

In the present study, proportions of oocytes that resumed meiosis (GVBD–MII) were around 60–70% within 30 h in the ligated oviduct: these rates are similar to those reported by Hewitt and England (1999b) after 96 h of culture (68%) and Bogliolo et al. (2002) after 48 and 72 h of maturation (60 and 58.1%). Moreover, proportions of oocytes that reached MI–MII stages in the ligated oviduct ranged between 12.5 and 31.9%, after only 30 h of culture. These results suggest that the intact oviduct in which oocytes were cultured promotes meiotic maturation that is resumed in a shorter time interval compared with what has been reported by other authors.

The isolated oviductal tract of the oviduct may provide an in vitro environment with characteristics which diverge from a monolayer of oviductal cells or an exposed mucosa, as in the open oviduct used in the present study. In fact, cultured cells are represented by a limited number of cell types (Hewitt and England 1999b), while in the intact oviduct all cellular types are present. Moreover, the spatial arrangement and the physical contact between mucosa and the oocytes create a microenvironment different from the conditions that are provided in vitro by culture drops. It must also be stressed that in the present study, a higher number of oocytes (up to 20) were cultured in the single oviductal tract than in the drop. As reported by Otoi et al. (2002) there is an influence of oocyte culture density on meiotic competence of canine oocytes. This suggests that a further physical variable renders the cultural conditions in the ligated oviduct different from the one in the drop and that it must be taken into account for the definition of an in vitro system for canine oocytes.

In the present study the high percentage of oocytes that resumed meiosis was mainly because of the increase of GVBD to DK stages. These data may also indicate that 30 h of culture of the oocytes in the oviduct supported the resumption of meiosis, but were not sufficient to complete nuclear maturation. However, the extension of the culture time in the oviduct up to 48 h, increased oocyte and oviductal cell degeneration.

The prevalence of GVBD–DK stages and the variability of the MI–MII rates between two experiments of the present study could be caused by the undefined conditions represented by a co-culture with oviductal tissue.

In the present work, oocytes and oviducts were collected from bitches at random stages of the oestrous cycle, however, the influence of the reproductive status of the donor bitch on in vitro maturation of the oocytes is still controversial. It has been reported that some form of pre-ovulatory priming are required for canine oocytes to become receptive to oviductal factors stimulating resumption of meiosis and full maturation. Yamada et al. (1993) reported that oocytes from anoestrous bitches showed no tendency to resume meiosis and a similar effect of the phase of the oestrous cycle when investigating the relation between the presence of oocytes–cumulus cell communications and meiotic competence was described (Luvoni et al. 2001). It is also well-known that the secretory activity of the oviductal cells changes in relation to the phase of the oestrous cycle and this may explain why the proportions of oocytes that reached MI–MII stages were mostly represented by MII oocytes in the work of Bogliolo et al. (2002) in which oviductal cells used for the co-culture were collected from bitches at this stage of the cycle. Conversely, in a recent work (Rodrigues and Rodrigues 2003b) no influence of the reproductive status of the female has been observed on the in vitro maturation rates of canine oocytes.

In conclusion, the results of the present study demonstrate that the culture of canine oocytes in the isthmus–ampullar tract of the isolated oviduct positively influences the oocyte survival and progression of nuclear maturation that is resumed within 30 h of culture.

The culture in the isolated oviduct is far from being a practical and defined culture system, but it suggests that other factors are involved in the maturation and viability of canine oocytes besides the presence of oviductal cells.

Oocyte maturation is affected by both the oviductal fluid composition and the physical contact with the oviductal mucosa and further studies are advisable in order to investigate other oviductal factors involved in the mechanism of resumption and progression of meiosis and in order to standardize a defined in vitro system for canine oocyte maturation.

Acknowledgements

This project was funded by the Ministero dell'Istruzione, dell'Università e della Ricerca, Italia (FIRST). The authors wish to thank Dr Silvia Modina for the histological analysis of the oviductal tissue, and all cooperating veterinary clinics and also the colleagues of the department for generously providing bitch ovaries.