In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers

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Theriogenology 77 (2012) 347–355 www.theriojournal.com

In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers Mohammed Ali Abdel-Ghania,c, Takashi Shimizub, Tomoyoshi Asanoa,c, Hiroshi Suzukia,c,* a

Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, 080-8555, Japan b Animal Reproduction Science, Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, 080-8555, Japan c The United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan Received 27 October 2010; received in revised form 21 July 2011; accepted 3 August 2011

Abstract The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO2, 5% O2, and 90% N2. All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 ␮g/mL of estradiol-17␤, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 ␮M of ␤-mercaptoethanol, 100 IU/mL of penicillin, and 100 ␮g/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P ⬍ 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P ⬎ 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P ⬍ 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P ⬍ 0.05), and least oocyte degeneration (P ⬍ 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes. © 2012 Elsevier Inc. All rights reserved. Keywords: Granulosa cell monolayers; IVM; IVP; Canine oocytes; Dog

1. Introduction Assisted reproductive techniques (ARTs) including IVM of oocytes are eminently desirable for rescuing genetic materials from individuals that fail to repro* Corresponding author: Tel.: ⫹81-155-49-5640; fax: ⫹81-155-495643. E-mail address: [email protected] (H. Suzuki). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.08.007

duce. These techniques are also valuable in improving the genetic management of rare populations maintained ex situ as an insurance for counterparts living in nature [1]. However, despite recent tremendous advances in the application of ARTs to other domestic animals, consistent and controlled reproduction either by natural or assisted breeding has remained elusive in canids. This is mostly due to their unique reproductive characteristics, including polyovulation, non-seasonal repro-

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ductive cycle, ovulation of immature oocytes at the germinal vesicle stage, and a 48 to 72 h period of postovulatory oocyte maturation in the oviduct. Therefore, a combination of these factors make it difficult to develop ARTs for canids [2,3]. The major challenge in developing IVM systems is creation of environmental conditions that can support oocyte development and would resemble an in vivo situation. Many researchers have examined the feasibility of IVM of canine oocytes [4 – 8]. Other researchers have also attempted to add oviductal epithelial cells [9], synthetic oviductal fluid (SOF) [10], mouse embryonic fibroblasts (MEF), and canine embryonic fibroblasts (CEF) [11] to the oocyte culture medium to address the above problems. In other studies, concentrated materials of protein sources [8] including gonadotrophins [12] and growth factors [13,14] have also been used. Nevertheless, the rates of maturation of canine oocytes to metaphase-II (M-II) remain low (⬍ 25%), especially when compared with those of many other mammalian species. For example, maturation rates of 90% have been achieved in cattle [15], and an 87% rate was attained in sheep [16]. Other reported successful maturation rates included 88% for pigs [17], 93% for mice [18], and 70% for cats [19]. The acquisition of developmental competence of oocytes is a limiting step that determines the ability of the oocyte to undergo successful fertilization and embryonic development. Furthermore, the inability to develop a consistent and an effective IVM system has resulted in limited success rates in IVF and IVC [20]. The final differentiation of the oocyte is orchestrated by a complex network of growth factors and cytokines leading to proper nuclear and cytoplasmic maturation. There is evidence that supplementation of IVM medium with granulosa cells improves nuclear and cytoplasmic maturation of oocytes in cattle [21], sheep [22], goats [23], camels [24], and monkeys [25], as well as the incidence of normal fertilization [26]. Since granulosa cells are found in vivo within developing follicles and undoubtedly play an important role in the oocyte maturation process, the present study was designed to investigate the influence of granulosa cell monolayers on the nuclear maturation of canine oocytes with and without cumulus cells during IVM. 2. Materials and methods 2.1. Assessment of reproductive status of the donors The reproductive status of the donors was categorized as follows [27]: (a) anestrus, when the ovaries had

no follicles or pronounced luteal tissues; (b) estrus (follicular phase), when one or more visible follicles were present; and (c) diestrus (luteal phase), when one or more evident corpora lutea were present. 2.2. Collection and preparation of cumulus-oocyte complexes (COCs) Ovaries were collected from healthy domestic bitches undergoing routine ovariohysterectomy in local veterinary clinics. The animals (n ⫽ 42) were of various breeds, ranging in age from 8 mo to 7 yr. Both ovaries from each bitch were transported to the laboratory within 1 h after collection, in a thermo flask containing physiological sterile saline supplemented with 100 IU/mL of penicillin (Calbiochem, Inc., La Jolla, CA, USA) at 37 °C. After transportation, the fat, ligaments, and medulla were carefully trimmed off and removed. The COCs were released by repeatedly slicing the ovarian cortex with a scalpel blade (Feather, Osaka, Japan) at 37 °C. These COCs were placed in 35 mm petri dish (Falcon # 3001; Becton Dickinson, Lincoln Park, NY, USA) containing PB1 medium [28] supplemented with 3 mg/mL of BSA (Sigma, St. Louis, MO, USA) and 100 ␮g/mL of streptomycin (MEIJI Co., Tokyo, Japan), and examined under a dissecting microscope (SMZ1500, Nikon Instech Co., Ltd. Tokyo, Japan). After three washes in the same medium, the COCs were selected by observation under an inverted microscope (DMIR/E, LEICA Co., Wetzlar, Germany) according to previously described criteria [29]. The parameters were those reported to favor meiotic competence, which included the uniformity of ooplasm, homogeneous dark cytoplasm with more than three layers of compact cumulus cells and oocytes ⬎ 110 ␮m in diameter. The vitelline diameter of all COCs was measured with a calibrated ocular micrometer. 2.3. Preparation of cumulus-free oocytes Cumulus-free oocytes were prepared by exposing the selected COCs to 0.2% of hyaluronidase (Sigma) for 15 min with gentle pipetting to remove cumulus cells. 2.4. Preparation of bovine granulosa cell monolayers (BGML) Bovine granulosa cells were prepared as described by Maeda et al [21]. Briefly, bovine ovaries were collected from a local abbatoir and transported to the laboratory in a thermo flask. The granulosa cells were obtained by aspiration of a small antral follicle (2 to 5 mm in diameter) with an 18 ga needle. The follicular

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fluid containing granulosa cells was centrifuged at 180 ⫻ g for 5 min. The resulting pellet of granulosa cells was washed twice by centrifugation at 180 ⫻ g for 5 min with 20 mL of calcium and magnesium free PBS (WAKO, Tokyo, Japan) containing 100 IU/mL of penicillin and 100 ␮g/mL of streptomycin. The pellet was then suspended in 10 mL of 0.1% hyaluronidase solution for 10 to 20 min at 38.5 °C. Cells were pipetted vigorously to encourage separation of the cells and then washed twice by centrifugation at 180 ⫻ g for 5 min with 10 mL of calcium and magnesium free PBS containing antibiotics. After the final washing, cells were resuspended in 20 mL of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma), 100 IU/mL of penicillin, 100 ␮g/mL streptomycin, and 1% amphotricin (Sigma). The final concentration of cells was adjusted to 25 ⫻ 105 cells/mL (counted with a haemocytometer) by adding culture medium. Thereafter, 2 mL of suspension was then placed in a collagen type I-coated multiwell dish (35 mm, IWAKI, Tokyo, Japan) and cultured at 38.5 °C in a humidified incubator with 5% CO2 in air for 3 to 5 d to produce a confluent monolayer. Cell viability was assessed by mixing 2 ␮L of the granulosa cell suspension with 2 ␮L of 0.4% trypan blue (GIBCO, Invitrogen Corporation, Grand Island, NY, USA) for 2 min. The percentage of viable cells was ⬎ 80% at the start of each culture. 2.5. Preparation of canine granulosa cell monolayers (CGML) Canine granulosa cells were prepared in the same manner as BGML samples. However, these granulosa cells were obtained by repeatedly slicing the ovarian cortex with a scalpel blade at 37 °C, and then the COCs were suspended in 0.2% of hyaluronidase for 30 to 40 min at 38.5 °C to disperse the granulosa cells. 2.6. In vitro maturation Intact or denuded oocytes were cultured in DMEM either with or without BGML (25⫻105 cells/ mL) or CGML (25⫻105 cells/ mL). All media were supplemented with 10% of FCS, 50 ng/mL of EGF (Sigma), 2 ␮g/mL of estradiol-17␤ (Sigma), 0.1 IU/mL of hCG (Sankyo, Tokyo, Japan), 0.1 IU/mL of FSH (Sigma), 0.25 mM of pyruvic acid (Wako, Ltd. Tokyo, Japan), 100 ␮M of ␤-mercaptoethanol (Sigma), 100 IU/mL of penicillin, and 100 ␮g/mL of streptomycin. Oocytes were randomly allocated into six treatment groups:

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Group 1 (control group): oocytes enclosed by cumulus cells were cultured in DMEM. Group 2 (co-culture with CGML group): oocytes enclosed by cumulus cells were co-cultured in DMEM with CGML. Group 3 (co-culture with BGML group): oocytes enclosed by cumulus cells were co-cultured in DMEM with BGML. Group 4 (denuded control group): denuded oocytes were cultured in DMEM. Group 5 (denuded co-culture with CGML group): denuded oocytes were co-cultured in DMEM with CGML. Group 6 (denuded co-culture with BGML group): denuded oocytes were co-cultured in DMEM with BGML. In all experimental groups, 20 to 25 oocytes were incubated in 500 ␮L of medium in a 35 mm petri dish covered with mineral oil (Nacalai Tesque, Inc. Kyoto, Japan) at 38.5 °C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 72 h. 2.7. Assessment of cumulus expansion After 24, 48, and 72 h of incubation, the effects of culture condition on cumulus expansion were assessed by stereomicroscope examination, taking into account mucification when the oocytes were surrounded by completely dispersed cumulus cells [30]. 2.8. Assessment of nuclear status Cumulus cells of oocytes were removed by exposure to 0.2% of hyaluronidase for 15 min with gentle pipetting. The denuded oocytes were fixed and permeabilized in PBS containing 3.7% (w/v) of paraformaldehyde (WAKO) for 15 min. Then, they were washed three times in PB1 supplemented with 3 mg/mL of BSA. The cumulus-free oocytes were stained with 10 ␮g/mL of propidium iodide (Sigma) in PBS containing 0.1% of polyvinyl alcohol (Sigma) and incubated for 15 min in darkness. Afterwards, they were washed three times in PB1, placed on glass slides, and overlaid with a coverslip. The chromatin state was evaluated under a fluorescence microscope with UV light (C-SHG1, Nikon Co., Tokyo, Japan) to determine the meiotic stage according the study of De los Reyes et al [30]. The stages were as follows: (a) immature or germinal vesicle (GV, Fig. 1A), when the nucleolus was surrounded by condensed chromatin; (b) resumption of meiosis or germinal vesicle break down (GVBD, Fig. 1B), when the chromatin was dispersed; (c) metaphase I (M-I, Fig. 1C), when chromosomes were highly compact in a metaphasic plate and migrating to the poles; (d) metaphase II; (M-II, Fig. 1D), when chromosomes were in second metaphase with extrusion of the first polar body; (e) degenerated (deg, Fig. 1E), when

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Fig. 1. Fluorescence photomicrographs (⫻ 400) of canine oocytes stained with propidium iodide showing chromatin configuration: (A) Germinal vesicle: condensed chromatin (arrow); (B) Germinal vesicle break down: chromatin was dispersed (arrow); (C) Metaphase I: chromosomes were compact in a metaphasic plate and migrating to the poles (arrow); (D) Metaphase II: the extrusion of the first polar body (arrow) (E) Degenerated: oocytes displayed loss of membrane integrity or dispersed chromosomes (arrow). (F) Unclassified: oocytes without identifiable chromatin.

oocytes displayed loss of membrane integrity or dispersed chromosomes; (f) oocytes with unidentifiable chromatin were counted as unclassified (Fig. 1F). All experiments were carried out in accordance with the guidelines for the care and use of animals approved by Obihiro University of Agriculture and Veterinary Medicine. 2.9. Statistical analysis Oocytes were randomly allocated to experimental groups in 10 experimental replicates. The proportion

of oocytes with expanded cumulus cell (mucification) and the proportion of those reaching each stage of nuclear maturation in each treatment group were subjected to arcsine transformation, and evaluated by ANOVA (Statistical Analysis System, SPSS, Chicago, IL, USA), followed by posthoc multiple comparisons using the least significant difference (LSD) test. The results were expressed as mean ⫾ SD. All differences were considered significant at a confidence level of P ⬍ 0.05.

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Table 1 Effects of granulosa cell monolayer on mean (⫾SD) percentage of canine oocytes with cumulus cell expansion following in vitro culture for varying intervals. Stage of COCs donor

Anestrus

Estrus and/or diestrus

Treatment

DMEM CGML BGML DMEM CGML BGML

No. oocytes examined

Oocytes with cumulus expansion (%)

108 106 112 112 111

24 h

48 h

72 h

4.7 ⫾ 0.0a 10.0 ⫾ 0.5b 16.9 ⫾ 0.0c 6.0 ⫾ 0.2a 15.8 ⫾ 0.1c 17.8 ⫾ 0.2c

19.6 ⫾ 0.4a 38.8 ⫾ 0.0b 56.0 ⫾ 0.6c 27.7 ⫾ 0.0d 53.5 ⫾ 0.0c 57.8 ⫾ 0.2c

36.9 ⫾ 0.0a 61.3 ⫾ 0.2b 79.4 ⫾ 0.4c 41.1 ⫾ 0.1a 71.4 ⫾ 0.0d 83.0 ⫾ 0.1c

CGML, canine granulosa monolayer; BGML, bovine granulosa monolayer. a⫺d Within a column, means without a common superscript differed (P ⬍ 0.05).

3. Results 3.1. Cumulus cell expansion The presence of BGML and CGML in the culture medium enhanced (P ⬍ 0.05) the incidence of cumulus cell expansion of COCs after 24, 48, and 72 h of incubation, irrespective of the estrous cycle. The maximum percentage (P ⬍ 0.05) of oocytes with cumulus expansion occurred in BGML at 72 h after incubation (Table 1). 3.2. Effect of CGML and BGML on nuclear maturation of canine oocytes enclosed by cumulus cells In oocytes retrieved from ovaries at estrus and/or diestrus, as shown in Table 2, the highest percentage of M-II oocytes (P ⬍ 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In oocytes collected from ovaries at anestrus, the percentage of M-II oocytes was highest (P ⬍ 0.05) in the BGML group (17.9%), whereas lower percentages were in both the CGML group (3.6%) and the control group (2.8%, Table 2). Oocytes from estrous and/or diestrous bitches had a higher rate of maturation to M-II (27.0%) than those from anestrous bitches (17.9%; P ⬍ 0.05). The overall meiotic resumption was greater (P ⬍ 0.05) in the BGML group than in both the CGML group and control group. In addition, treatment with BGML resulted in less (P ⬍ 0.05) oocyte degeneration than the other experimental groups. 3.3. Effect of CGML and BGML on nuclear maturation of cumulus-free oocytes In cumulus-free oocytes collected from estrous and/or diestrous ovaries (Table 3), the proportions of M-II oocytes co-cultured with the CGML were low

(3.0%) and similar (P ⬎ 0.05) to control (3.0%). However, co-cultivation with BGML improved (P ⬍ 0.05) the ability of denuded oocytes to develop to M-II (10.2%). The presence of BGML impaired (P ⬍ 0.05) the incidence of oocyte degeneration compared to the other experimental groups. In cumulus-free oocytes recovered from anestrous ovaries, percentages of M-II oocytes were higher (P ⬍ 0.05) in the BGML (8.2%) than in both the CGML (3.2%) and the control (3.1%). The presence of BGML positively influenced (P ⬍ 0.05) the overall meiotic resumption regardless the estrous cycle. 4. Discussion In the present study, there was a significant increase in maturation rates of canine oocytes co-cultured with the BGML. The proportion of oocytes that resumed meiosis was 27.0%, which seemed greater than that reported by previous studies (⬍ 25%) [4 – 8]. In primary follicles containing growing oocytes, the surrounding granulosa cells start to proliferate, and at the time of antrum formation, two specific populations of granulosa cells could be distinguished: 1) cumulus granulosa cells, which enclose the oocyte with the corona cells as the innermost layers; and 2) mural granulosa cells which line the follicular wall [31]. Cumulus and mural granulosa cells together with the oocyte form a gap junction mediated syncytium, which is essential for oocyte growth to proceed, and granulosa cells supply the oocytes with nutrients and connect them to the external world [32]. The influence of BGML is probably mediated through various meiosis promoting factors produced by bovine granulosa cells, including EGF, activin, transforming growth factor-beta (TGF␤), and basic fibroblast growth factors (bFGF) [33,34]. There is a substantial body of research, which has

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Table 2 Mean (⫾SD) percentage of cumulus enclosed canine oocytes at various meiotic stages following co-culture with granulosa cell monolayers for 72 h. Stage of COCs donor

Anestrus

No. oocytes examined

DMEM CGML BGML DMEM CGML BGML

106 108 106 112 112 111

Meiotic stage (%) GV

GVBD

MI

MII

Degenerate

Unclassified

GVBD-MII

26.2 ⫾ 0.7a 23.9 ⫾ 0.1a 19.5 ⫾ 0.3a 25.3 ⫾ 1.0a 25.4 ⫾ 0.6a 9.9 ⫾ 0.5b

14.2 ⫾ 1.5ab 19.0 ⫾ 0.3ab 23.0 ⫾ 1.1bc 11.1 ⫾ 0.6a 23.1 ⫾ 0.5bc 31.4 ⫾ 2.8c

7.3 ⫾ 0.2a 8.3 ⫾ 1.0a 17.8 ⫾ 0.3b 8.9 ⫾ 1.1a 14.0 ⫾ 0.2ab 17.2 ⫾ 1.7b

2.8 ⫾ 2.4a 3.6 ⫾ 1.3a 17.9 ⫾ 0.0b 3.5 ⫾ 1.2a 7.9 ⫾ 0.1a 27.0 ⫾ 0.7c

35.9 ⫾ 1.5a 19.4 ⫾ 0.7bc 12.0 ⫾ 0.4cd 28.3 ⫾ 1.3ab 19.5 ⫾ 0.9bc 7.7 ⫾ 0.3d

9.6 ⫾ 0.5a 22.8 ⫾ 1.3b 8.4 ⫾ 0.6a 19.3 ⫾ 0.9b 8.4 ⫾ 0.5a 2.8 ⫾ 0.8c

26.7 ⫾ 1.1a 32.2 ⫾ 1.9a 59.5 ⫾ 0.2b 24.8 ⫾ 2.0a 45.6 ⫾ 0.3c 76.9 ⫾ 0.6d

CGML, canine granulosa monolayer; BGML, bovine granulosa monolayer; GV, germinal vesicle; GVBD, germinal vesicle break down; MI, metaphase I; MII, metaphase II. a⫺d Within a column, means without a common superscript differed (P ⬍ 0.05).

Table 3 Mean (⫾SD) percentage of denuded canine oocytes at various meiotic stages following co-culture with granulosa cell monolayers for 72 h. Stage of COCs donor

Anestrus

Estrus and/or diestrus

Treatment

No. oocytes examined

DMEM CGML BGML DMEM CGML BGML

105 105 109 108 106 107

Meiotic stage (%) GV

GVBD

MI

MII

Degenerate

Unclassified

GVBD-MII

22.8 ⫾ 0.2ab 26.7 ⫾ 0.1a 18.9 ⫾ 0.3b 20.4 ⫾ 0.1b 24.7 ⫾ 0.2ab 27.2 ⫾ 0.9a

7.4 ⫾ 0.2a 18.0 ⫾ 0.9bc 18.4 ⫾ 0.2bc 12.2 ⫾ 0.5ab 20.0 ⫾ 0.4c 25.0 ⫾ 0.5c

8.3 ⫾ 0.6a 5.5 ⫾ 2.0a 15.4 ⫾ 0.3b 8.0 ⫾ 0.5a 7.3 ⫾ 0.7a 15.5 ⫾ 0.3b

3.1 ⫾ 1.0a 3.2 ⫾ 1.1a 8.2 ⫾ 0.1b 3.0 ⫾ 1.0a 3.0 ⫾ 0.9a 10.2 ⫾ 1.3b

30.9 ⫾ 0.6a 13.6 ⫾ 1.8bc 17.5 ⫾ 0.0b 30.2 ⫾ 0.4a 20.3 ⫾ 0.5b 9.0 ⫾ 0.3c

25.6 ⫾ 0.2a 28.8 ⫾ 0.9a 23.0 ⫾ 0.4a 25.8 ⫾ 0.2a 22.8 ⫾ 0.7a 10.9 ⫾ 0.6b

20.0 ⫾ 0.6a 28.3 ⫾ 2.7ab 42.2 ⫾ 0.8cd 22.9 ⫾ 0.9ab 32.4 ⫾ 1.3bc 47.7 ⫾ 0.7d

CGML, canine granulosa monolayer; BGML, bovine granulosa monolayer; GV, germinal vesicle; GVBD, germinal vesicle break down; MI, metaphase I; MII, metaphase II. a⫺d Within a column, means without a common superscript differed (P ⬍ 0.05).

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Estrus and/or diestrus

Treatment

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introduced the concept that these growth factors stimulate the IVM of oocytes in rats [35], mice [36], cattle [37], humans [38], and pigs [39]. It is likely that such factors are not species-specific, and most intriguing was what appeared to be diffusible/paracrine factors from BGML that led to the beneficial effect of BGML coculture on oocyte meiotic maturation in this study. However, there is currently no information available on secretion of these meiosis-promoting factors by canine granulosa cells. Interestingly, bovine granulosa cells can secrete high concentrations of estrogen and progesterone when supplemented with either FCS or BSA [40]. Moreover, positive effects of estrogen and progesterone on IVM of oocytes have been demonstrated in several mammalian species. Similar results also have been reported in domestic dogs. Culture of canine oocytes supplemented with either estrogen or a combination of estrogen and progesterone promoted resumption of meiosis and enhanced development to M-II stage in vitro [41]. Furthermore, bovine granulosa cells were involved in mediating the stimulatory effects of estrogen and EGF on nuclear maturation of oocytes, and estrogen and EGF are emerging as critical elements to the acquisition of full oocyte competence [42]. Abeydeera et al [43] suggested that granulosa cells are important for increase the concentration of intracellular glutathione during the IVM of oocytes. This has an important role in protecting cells against oxidative stress and is correlated with nuclear and cytoplasmic maturation. It is not known whether canine granulosa cells secrete either estrogen or progesterone, but presumably one of these factors, or several of them acting in concert, secreted from bovine granulosa cells induced nuclear maturation of canine oocytes. Moreover, the significant lower rate of maturation to the M-II stage for oocytes co-cultured in CGML than those in BGML may be attributed to the condition in which the cells were harvested. Unlike canine granulosa cells, bovine granulosa cells were retrieved by aspiration of small antral follicles, which were probably steroidogenically competent with pure granulosa cells. However, the canine granulosa cells were obtained by ovarian slicing, which could introduce minor, but unwanted, trace impurities such as thecal and interstitial cells, as well as cells from immature follicles in addition to granulosa cells from mature follicles. It appears that a combination of these factors could not support the meiotic maturation rate, as observed in the present study.

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In cumulus-enclosed oocytes, the rate of meiotic resumption was higher than that of cumulus-denuded oocytes, regardless of stage of estrous cycle (Tables 2 and 3). This was expected, since previous studies with various species have shown that the surrounding cumulus cells have an important role in the oocyte meiotic maturation process [32]. The role of cumulus cells in canids may be more important than in other species [44], because the multilayered cumulus cell mass remains closely attached around oocyte up to the morula stage [45]. The potential role of cumulus cells has been the subject of several reviews, all of which suggest that these cells were intimately connected with the oocyte through long microvilli that traverse through the zona to contact the oolemma, so as to form gap junctions and desmosomes [46]; one of the routes by which the cumulus cells transmit factors to the oocyte is gap junctional communication (GJC) [47]. Thus, the GJC between cumulus cells and the oocytes plays an important role in the transmission of meiosis-activating components and some low molecular substrates such as ions, nucleotides, and amino acids [48]. In the present study, rates of maturation to the M-II stage for oocytes from bitches at estrus and/or diestrus were significantly higher than those from bitches at anestrus (Tables 2 and 3). However, published information on the relationship between estrous cycle and the nuclear maturation of oocytes are contradictory. Some investigators have indicated no association [49,50], whereas others have shown that estrous cycle stage significantly impacts developmental capacity of the oocytes [44]. Willingham-Rocky et al [51] similarly reported that oocytes obtained from estrous bitches were more likely to develop to the M-II stage than oocytes obtained from anestrous bitches. Moreover, the oocytes recovered from diestrous bitches were as likely to achieve M-II in vitro as those from bitches at estrus (Tables 2 and 3). Differences in meiotic competence of oocytes across stages of the estrous cycle are most likely due to the influence of the estrous cycle stage on the functional status of gap junctions between cumulus cells and oocytes. Furthermore, the presence and persistence of cumulus– oocyte communications in COCs is correlated to the ability of the oocytes to resume meiosis [52], and there is a lack of communication between the ooplasm and cumulus cells of oocytes obtained from anestrous bitches [44]. Additionally, the higher percentage of meiotic progress observed in the oocytes obtained from estrous ovaries can be attributed to the exposure of these oocytes to a follicular environment enriched by estradiol, progesterone, and other

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unknown factors [53]. Alternatively, it may be as a result of either early or mid-atresia of follicles that have lost their ability to inhibit acquisition of meiotic competence by the enclosed oocytes [54]. This was consistent with our data in which the estrous cycle had a significant effect on the proportion of oocytes maturing to M-II stage in culture. In conclusion, the present study demonstrated that using BGML for IVM procedure enhanced meiotic resumption of canine oocytes. Although our study indicates that BGML co-culture can promote the nuclear maturation of denuded oocytes during IVM, the importance of cumulus cells cannot be neglected. Furthermore, the stage of the estrous cycle during oocyte collection was one of the key steps towards successful nuclear maturation. Acknowledgments We thank Dr. Aboge Gabriel Oluga for his helpful advice and for editing the paper.

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