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Association of distributions of three types of germinal vesicle stage oocytes with the canine follicle location in the ovary

Published online by Cambridge University Press:  21 July 2010

L.X. Wang ,
S. Wang ,
J. Hou ,
R.L. Hu  and
X.R. An
L.X. Wang
Affiliation:
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
S. Wang
Affiliation:
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
J. Hou
Affiliation:
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094, China.
R.L. Hu
Affiliation:
Institute of Veterinary Medicine Academy of Military Medical Science of PLA, Changchun 130062, China.
X.R. An*
Affiliation:
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuan-ming-yuan West Road, Beijing 100094, China.
*
All correspondence to: X.R An, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, No. 2 Yuan-ming-yuan West Road, Beijing 100094, China. Tel: +86 10 627333 5522. Fax: +86 10 62733463. e-mail: wlxue@163.com
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Summary

The objective of current study was to compare the nuclear configurations of canine oocytes recovered from between follicles after isolation. Follicles isolated were classified into follicle-S (follicles located in the ovarian surface) and follicle-I (follicles located inside the ovary) based on the follicle location in the ovary. Nuclear stages of canine oocytes recovered from follicle-S and follicle-I were examined by phase-contrast microscopy after isolation. Results demonstrated that canine GV stage oocytes can be classified into three types based on the status of the nuclear envelope, nucleolus, and chromatin: type A, type B, and type C. In follicle-S group, the majority (95.5%) of canine GV stage oocytes was of type B. All canine GV stage oocytes recovered from follicle-S (including type B and type C) were characterized by nuclear envelope disappearance prior to nucleolus collapse. In contrast, in follicle-I group, the majority (60.2%) of canine GV stage oocytes was of type C. Unexpectedly, a small proportion of canine GV stage oocytes from follicle-I (donated type A) were characterized by nuclear envelope disappearance following nucleolus collapse. In conclusion, nuclear configurations of each type of canine GV stage oocytes may differ from each other. Distributions of each type of canine GV stage oocytes may associate with the follicle location in the ovary.

Keywords

Canine oocytesGV stageNuclear envelopeNucleolusFollicle-S
Type
Research Article
Information
Zygote , Volume 19 , Issue 1 , February 2011 , pp. 91 - 95
Copyright
Copyright © Cambridge University Press 2010

Introduction

In canine oocytes, in vitro maturation (IVM) is characterized by low rates of maturation to metaphase II (MII) (Mahi & Yanagimachi, Reference Mahi and Yanagimachi1976; Yamada et al., Reference Yamada, Shimazu, Kawaji, Nakazawa, Naito and Toyoda1992, Reference Yamada, Shimazu, Kawano, Nakazawa, Naito and Toyoda1993; Nickson et al., Reference Nickson, Boyd, Eckersall, Ferguson, Harvey and Renton1993; Bolamba et al., Reference Bolamba, Borden-Russ and Durrant1998; Hewitt & England, Reference Hewitt and England1998, Reference Hewitt and England1999; Metcalfe, Reference Metcalfe1999; Otoi et al., Reference Otoi, Fujii, Tanaka, Ooka and Suzuki2000a, Reference Otoi, Fujii, Tanaka, Ooka and Suzuki.b, Reference Otoi, Ooka, Murakami, Karja and Suzuki2001, Reference Otoi, Willingham, Shin, Kraemer and Westhusin2002; Saint-Dizier et al., Reference Saint-Dizier, Renard and Chastant-Maillard2001; Songsasen et al., Reference Songsasen, Yu and Leibo2002; Hatoya et al., Reference Hatoya, Sugiyama, Torii, Wijewardana, Kumagai, Sugiura, Kida, Kawate, Tamada, Sawada and Inaba2006). However, the actual reason for the low IVM rates in canines remains unclear. It has been acknowledged that canine oocytes are spontaneously ovulated at the germinal vesicle (GV) stage (Yamada et al., Reference Yamada, Shimazu, Kawaji, Nakazawa, Naito and Toyoda1992; Reynaud et al., Reference Reynaud, Fontbonne, Marseloo, Thoumire, Chebrout, Viaris de Lesegno and Chastant-Maillard2005). After ovulation, they require an extended time in the oviducts to complete the second meiotic maturation (Concannon, Reference Concannon1994). In most mammalian animals, oocyte maturation occurs within ovarian follicles.

A previous study was conducted to assess the in vivo developmental competence of oocytes and embryos (Reynaud et al., Reference Reynaud, Fontbonne, Marseloo, Thoumire, Chebrout, Viaris de Lesegno and Chastant-Maillard2005). However, no canine oocytes at the germinal vesicle breakdown (GVBD) stage were detected in their study. Probably, canine oocytes need such a short time in vivo to develop from GV stage to metaphase I (MI) that oocytes at the GVBD stage have not been observed yet in such studies. Another study used super-ovulated canines to aspirate oocytes from follicles for in vitro maturation (Yamada et al., Reference Yamada, Shimazu, Kawaji, Nakazawa, Naito and Toyoda1992). This group also did not find any oocytes at the GVBD stage in their study. Therefore, these two studies reveal that canine oocytes recovered by follicle aspiration or in the oviduct require a very short time to progress beyond the GVBD stage in the first meiosis. In most other studies, canine oocytes with various nuclear stages (including GVBD stage) can be observed before or after in vitro maturation (De Los Reyes et al., Reference De Los Reyes, De Lange, Miranda, Palominos and Barros2005), when the majority of oocytes for IVM were recovered from the follicles located inside the ovary at the abattoir. It is possible that the configurations of nuclear status of canine GV stage oocytes recovered from follicle-S differ from those of follicle-I.

The objective of this study was to compare the configurations of nuclear envelope, nucleolus, and chromatin in three types of canine GV stage oocytes recovered from between follicle-S and follicle-I.

Materials and Methods

Ovaries and oocytes collection

Ovaries obtained from adult bitches at a local abattoir were collected within 1 h of removal, and transported to the laboratory in a thermal bottle containing prewarmed physiological saline solution at 37°C within 3 h of removal. Follicles were classified into two types based on their location in the ovary: follicle-S if follicles located in the ovarian surface, and follicle-I if follicles located inside the ovary. Cumulus–oocyte complexes (COCs) from follicle-S and follicle-I were collected and the nuclear stages of these were examined by phase contrast microscopy.

Experimental design

Ovaries at pro-estrous and estrous stages with apparent follicles located in the ovarian surface (follicle-S) were selected. In each replicate, canine oocytes from follicle-S were first obtained by follicle aspiration. Subsequently, those from follicle-I from the same ovary were obtained by slicing the ovarian cortex with a scalpel blade. Morphologically degenerated oocytes (such as following zona pellucida rupture) both from follicle-S and follicle-I were discarded. Selected COCs were fixed and stained to determine the nuclear stages by phase contrast microscope.

Assessment of nuclear stage of maturation

After collection, oocytes were denuded, fixed in acetic acid: ethanol:chloroform (3:6:2, v:v:v) for 2 to 3 min prior to fixation with acetic acid:ethanol (1:3, v:v) for 24 to 48 h (Songsasen et al., Reference Songsasen, Yu and Leibo2002; De Los Reyes et al., Reference De Los Reyes, De Lange, Miranda, Palominos and Barros2005; Hatoya et al., Reference Hatoya, Sugiyama, Torii, Wijewardana, Kumagai, Sugiura, Kida, Kawate, Tamada, Sawada and Inaba2006). These oocytes were then stained with 1% (w/v) orcein-acetic (Sigma-Aldrich). Nuclear maturation stages were examined by phase contrast microscopy at ×400 magnification. We classified these nuclear stages as GV, GV breakdown (GVBD), metaphase I (MI), metaphase II (MII), and unidentified, according to the methods described previously (De Los Reyes et al., Reference De Los Reyes, De Lange, Miranda, Palominos and Barros2005).

Statistical analysis

The data for numbers and percentages of oocytes that progressed to various nuclear stages, and the distribution of three types of canine GV stage oocytes were analysed by SPSS 10.0 software. Due to the few numbers of canine oocytes recovered from follicle-S in each replicate, the significant differences in maturational rates between the treatment groups have not been compared in current studies.

Results

The majority of canine oocytes was at the GV stage after isolation both from follicle-S and follicle-I (Table 1). Meanwhile, a small proportion of oocytes had undergone the resumption of the meiosis in both the follicle-S and follicle-I groups.

Table 1 Per cent of canine oocytes at various nuclear stages recovered from follicle-S and follicle-I.

aFollicle-S, follicles located in the ovarian surface; follicle-I, follicles located inside the ovary.

bDE: degenerated oocytes, GV: germinal vesicle stage, GVBD: germinal vesicle breakdown, MI/TI: metaphase I/telophase I, MII: metaphase II.

GV stages were classified into three types based on the status of the nuclear envelope and nucleolus: type A, type B, and type C. In follicle-S group, the majority (95.5%) of canine GV stage oocytes was type B, which were characterized by nuclear envelope disappearance prior to nucleolus collapse (Fig. 1). There was no type A canine GV stage oocytes in this group (Table 2). In the follicle-I group, the majority of canine GV stage oocytes was type C. In addition, a small proportion of canine GV stage oocytes from follicle-I (donated type A) were characterized by nuclear envelope disappearance following nucleolus collapse (Fig. 1).

Figure 1 Representative pictures of three types of canine GV stage oocytes (magnification ×400). (a) Type A canine GV stage oocytes, the nuclear envelope was distinct and the nucleolus began to disappear. (b) Type A canine GV stage oocytes, the nuclear envelope was less distinct while the nucleolus completely disappeared. (c) Type B canine GV stage oocytes, the nuclear envelope disappeared and the nucleolus was visible. (d) Type B canine GV stage oocytes, the nuclear envelope has disappeared and the nucleolus has begun to collapse. (e) Type C canine GV stage oocytes, the nuclear envelope has disappeared, while the nucleolus is visible. (f) Type C canine GV stage oocytes, the nuclear envelope has disappeared while the nucleolus has begun to collapse.

Table 2 Per cent of various types of canine GV stage oocytes recovered from follicle-S and follicle-I.

aCanine oocytes were classified into three types based on the status of their nuclear envelope, nucleolus and chromatin.

Type A, characterized by nuclear envelope disappeared following the nucleolus collapsed completely. Chromatin was clearly seen in type A. Type A was further classified into three stages: A-I, A-II, A-III. A-I indicated that the nuclear envelope was intact and the nucleolus was clear. Chromatin was not observed at this stage; A-II indicated that the nuclear envelope was intact while the nucleolus collapsed. Chromatin was observed clearly at this stage; A-III indicated that the nuclear envelope began to disappear while the nucleolus collapsed completely. Chromatin was observed clearly at this stage.

Type B, characterized by nuclear envelope disappeared prior to the nucleolus collapse completely. Chromatin was not observed in type B. Type B one was further classified into three stages: B-I, B-II, B-III. B-I indicated that nuclear envelope was intact and the nucleolus was clear. Chromatin was not observed at this stage; B-II indicated that the nucleolus was clear while the nuclear envelope disappeared completely. Chromatin was not observed at this stage; B-III indicated that the nuclear envelope disappeared completely while the nucleolus began to collapse. Chromatin was not observed at this stage.

Type C, characterized by nuclear envelope disappeared prior to the nucleolus collapse completely. Chromatin was clearly seen in type C. Type C one was further classified into three stages: C-I, C-II, C-III. C-I indicated that nuclear envelope was intact and the nucleolus was clear. Chromatin was not observed at this stage; C-II indicated that the nucleolus was clear while the nuclear envelope disappeared completely. Chromatin was observed clearly at this stage; C-III indicated that the nuclear envelope disappeared completely while the nucleolus began to collapse. Chromatin was observed clearly at this stage.

Discussion

Unexpectedly, we find that the major types of canine GV stage oocytes from follicle-S (type B) and follicle-I (type C) are different. One possibility is that follicle-I with type B canine GV stage oocytes are predominantly recruited and become dominant follicles. If this assumption holds, type B canine GV stage oocytes may acquire a higher developmental capacity than other types. Therefore, the maturational abilities among three types of canine GV stage oocytes should be evaluated in future studies.

Type B and type C canine GV stage oocytes may be specific as they are characterized by nuclear envelope disappear prior to nucleolus collapse. They differ from those reported in other domestic animals, such as porcine (Christmann et al., Reference Christmann, Jung and Moor1994; Sun et al., Reference Sun, Liu, Yue, Ma and Tan2004), goat (Sui et al., Reference Sui, Liu, Miao, Yuan, Qiao, Luo and Tan2005), and sheep (Liu et al., Reference Liu, Sui, Wang, Yuan, Luo, Xia and Tan2006). As reported in porcine GV stage oocytes, they were characterized by nuclear envelope disappearance following nucleolus collapse (Motlik & Fulka, Reference Motlik and Fulka1976). They are consistent with type A canine GV stage oocytes in this study.

The majority of oocytes obtained from ovaries after collection is still at the GV stage and a few per cent of these have resumed meiosis. This finding is consistent with other reports (Mahi & Yanagimachi, Reference Mahi and Yanagimachi1976; Hewitt et al., Reference Hewitt, Watson and England1995; Bolamba et al., Reference Bolamba, Borden-Russ and Durrant1998). This situation further confirms that a few per cent of canine oocytes from ovaries have competence in resumption of meiosis. For instance, Bolama et al. (Reference Bolamba, Borden-Russ and Durrant1998) reported that 19% to 28% of dog oocytes had undergone GVBD and maximum reported chromosome condensation (GVBD-DK) before culture. Unexpectedly, Yamada et al. (Reference Yamada, Shimazu, Kawaji, Nakazawa, Naito and Toyoda1992) observed that all oocytes after isolation were at GV stage without in vitro culture. It was most likely that maturation rates after recovery associated with the ovaries at various stages of estrous cycles and the majority of oocytes were obtained mainly from ovaries at the non-estrus stage in their study.

In summary, nuclear configurations of three types of canine GV stage oocytes may differ from each other. Distributions of three types of canine GV stage oocytes may associate with the location of the follicle in the ovary.

Acknowledgements

We sincerely thank the workers at the abattoir in Chang Chun city for providing canine ovaries.

References

Bolamba, D., Borden-Russ, K.D. & Durrant, B.S. (1998). In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles. Theriogenology 49, 933–42.CrossRefGoogle ScholarPubMed
Christmann, L., Jung, T. & Moor, R.M. (1994). MPF components and meiotic competence in growing pig oocytes. Mol. Reprod. Dev. 38, 85–90.CrossRefGoogle ScholarPubMed
Concannon, P.W. (1994). Biology of gonadotrophin secretion in adult and prepubertal female dogs. J. Reprod. Fertil. 101, 221–5.Google Scholar
De Los Reyes, M., De Lange, J., Miranda, P., Palominos, J. & Barros, C. (2005). Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes. Theriogenology 64, 1–11.CrossRefGoogle ScholarPubMed
Hatoya, S., Sugiyama, Y., Torii, R., Wijewardana, V., Kumagai, D., Sugiura, K., Kida, K., Kawate, N., Tamada, H., Sawada, T. & Inaba, T. (2006). Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes. Theriogenology 66, 1083–90.CrossRefGoogle ScholarPubMed
Hewitt, D.A. & England, G.C.W. (1998). The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro. Theriogenology 49, 957–66.CrossRefGoogle ScholarPubMed
Hewitt, D.A. & England, G.C.W. (1999). Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro. Anim. Reprod. Sci. 55, 63–75.CrossRefGoogle ScholarPubMed
Hewitt, D.A., Watson, P.F. & England, G.C. (1995). Effect of concentration of serum on in vitro maturation of domestic bitch oocytes. J. Reprod. Fertil. 15, 66–7.Google Scholar
Liu, Y., Sui, H.S., Wang, H., Yuan, J.H., Luo, M.J., Xia, P. & Tan, J. (2006). Germinal vesicle chromatin configurations of bovine oocytes. Microsc. Res. Tech. 69, 799–807.CrossRefGoogle ScholarPubMed
Mahi, C.A. & Yanagimachi, R. (1976). Maturation and sperm penetration of canine ovarian oocytes in vitro. J. Exp. Zool. 196, 189–96.CrossRefGoogle ScholarPubMed
Metcalfe, S.S. (1999). Assisted reproduction in the bitch. Thesis for the degree of Master of Science, Monash University, Victoria, Australia: 160 pp.Google Scholar
Motlik, J. & Fulka, J. (1976). Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J. Exp. Zool. 198, 155–62.CrossRefGoogle ScholarPubMed
Nickson, D.A., Boyd, J.S., Eckersall, P.D., Ferguson, J.M., Harvey, M.J. & Renton, J.P. (1993). Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches. J. Reprod. Fertil. Suppl. 47, 231–40.Google ScholarPubMed
Otoi, T., Fujii, M., Tanaka, M., Ooka, A. & Suzuki, T. (2000a). Canine oocyte diameter in relation to meiotic competence and sperm penetration. Theriogenology 54, 535–42.CrossRefGoogle ScholarPubMed
Otoi, T., Fujii, M., Tanaka, M., Ooka, A. & Suzuki., T. (2000b). Effect of serum on the in vitro maturation of canine oocytes. Reprod. Fertil. Dev. 11, 387–90.CrossRefGoogle Scholar
Otoi, T., Ooka, A., Murakami, M., Karja, N.W.K. & Suzuki, T. (2001). Size distribution and meiotic competence of oocytes obtained from bitch ovaries at various stages of the oestrous cycle. Reprod. Fertil. Dev. 13, 151–5.CrossRefGoogle ScholarPubMed
Otoi, T., Willingham, L., Shin, T., Kraemer, D.C. & Westhusin, M. (2002). Effects of oocyte culture density on meiotic competence of canine oocytes. Reproduction 124, 775–81.CrossRefGoogle ScholarPubMed
Reynaud, K., Fontbonne, A., Marseloo, N., Thoumire, S., Chebrout, M., Viaris de Lesegno, C. & Chastant-Maillard, S. (2005). In vivo meiotic resumption, fertilization and early embryonic development in the bitch. Reproduction 130, 193–201.CrossRefGoogle ScholarPubMed
Saint-Dizier, M., Renard, J.P. & Chastant-Maillard, S. (2001). Induction of final maturation by sperm penetration in canine oocytes. Reproduction 121, 97–105.CrossRefGoogle ScholarPubMed
Songsasen, N., Yu, I. & Leibo, S.P. (2002). Nuclear maturation of canine oocytes cultured in protein-free media. Mol. Reprod. Dev. 62, 407–15.CrossRefGoogle ScholarPubMed
Sui, H.S., Liu, Y., Miao, D.Q., Yuan, J.H., Qiao, T.W., Luo, M.J. & Tan, J.H. (2005). Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species. Mol. Reprod. Dev. 71, 227–36.CrossRefGoogle ScholarPubMed
Sun, X.S., Liu, Y., Yue, K.Z., Ma, S.F. & Tan, J.H. (2004). Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes. Mol. Reprod. Dev. 69, 228–34.CrossRefGoogle ScholarPubMed
Yamada, S., Shimazu, Y., Kawaji, H., Nakazawa, M., Naito, K. & Toyoda, Y. (1992). Maturation, fertilization, and development of dog oocytes in vitro. Biol. Reprod. 46, 853–8.CrossRefGoogle ScholarPubMed
Yamada, S., Shimazu, Y., Kawano, Y., Nakazawa, M., Naito, K. & Toyoda, Y. (1993). In vitro maturation and fertilization of preovulatory dog oocytes. J. Reprod. Fertil. Suppl. 47, 227–9.Google ScholarPubMed
Figure 0

Table 1 Per cent of canine oocytes at various nuclear stages recovered from follicle-S and follicle-I.

Figure 1

Figure 1 Representative pictures of three types of canine GV stage oocytes (magnification ×400). (a) Type A canine GV stage oocytes, the nuclear envelope was distinct and the nucleolus began to disappear. (b) Type A canine GV stage oocytes, the nuclear envelope was less distinct while the nucleolus completely disappeared. (c) Type B canine GV stage oocytes, the nuclear envelope disappeared and the nucleolus was visible. (d) Type B canine GV stage oocytes, the nuclear envelope has disappeared and the nucleolus has begun to collapse. (e) Type C canine GV stage oocytes, the nuclear envelope has disappeared, while the nucleolus is visible. (f) Type C canine GV stage oocytes, the nuclear envelope has disappeared while the nucleolus has begun to collapse.

Figure 2

Table 2 Per cent of various types of canine GV stage oocytes recovered from follicle-S and follicle-I.