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Second polar body formation in rat ovarian oocytes matured in vitro
EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1976,6/l,
35-38. EXCERPTA MEDICA
Second polar body formation in rat ovarian oocytes matured in vitro G.H. Zeilmaker Department
and C.M.P.M.
of Endocrinology,
Verhamme
Growth and Reproduction,
Erasmus University, Rotterdam,
The Netherlands
ZEILMAKER, G.H. and VERHAMME, C.M.P.M. (1976): Second polar body formation in rat ovarian oocytes matured in vitro. Europ. J. Obstet. Gynec. reprod. Biol., 6/l,
35-38.
Denuded ovarian oocytes obtained from proestrous and prepuberal rats were incubated in medium containing lactate or without added energy source. Formation of a second polar body was observed about 24 h after the onset of culture. Formation by division of the fist polar body was excluded by observing second polar body formation in the presence of a degenerated first polar body. energy requirements; lactate; polar body degeneration
Introduction
Materials
and methods
Denuded rat ovarian oocytes will mature in vitro in media containing pyruvate or lactate and even in the absence of an added energy source. The first polar body is extruded 8-9 hours after explantation and often degenerates within several hours (Zeilmaker and Verhamme, 1974). It is well known that in the rat ovary the first polar body also degenerates soon after its formation even before follicle rupture (Odor, 1955; Zeilmaker, Vermeiden, Verhamme and Van Vliet, 1974). Formation of the second polar body in tubal oocytes can be induced by a variety of stimuli (Thibault, 1949; Austin and Braden, 1954) or by explantation of tubal oocytes in vitro (Zeilmaker and Verhamme, 1974). In the present study it was observed that in oocyte cultures which were regularly inspected, polar bodies were formed long after the expected time of first polar body formation. Data are presented which indicate that a second polar body may be formed in vitro in ovarian oocytes obtained from proestrous and prepuberal rats.
Ovarian oocytes were obtained from preovulatory follicles, dissected from 19 rats in proestrous (11 a.m., before expected LH surge) or by puncturing ovaries from twelve 30-day-old rats (3 p.m.). Cumulus cells were removed mechanically by pipetting and the washed oocytes obtained from one rat and containing a germinal vesicle were incubated under oil in a balanced salt solution (Biggers, Whittingham and Donahue, 1967) with 10 mM lactate or without added energy source. The cultures were gassed individually with 5% COz, 5% O2 and 90% N2 in a temperaturecontrolled room.
Results
Oocyte cultures were inspected for the formation of the first polar body 20-22 hours after explantation. At hourly intervals thereafter the number of oocytes with polar bodies was recorded. It appeared that of 62 oocytes isolated from pre35
36
G.H. Z&maker
and C.M.P.M. Verhamme:
Second polar body formed in rat ovarian oocytes
Fig. 1. Rat oocyte photographed twice during extrusion of a second polar body 24 h after explantation. During the interval between photographs (less than 5 mm) the oocyte turned clockwise about 30 degrees. Particles visible outside the zona pellucida near the degenerated polar body (top) are out of focus in the bottom photograph, and visible near the degenerated polar body through the zona.
G.H. Zeilmaker and C.M.P.M. Verhamme:
Second polar body formed in rat ovarian oocytes
Fig. 2. Rat oocyte with degenerated first polar body and a second polar body which has been fformed less than 2 h before photography.
Fig. 3. Rat oocyte with degenerated first polar body and fully formed second polar body.
31
38
G.H. Zeilmaker and C.M.P.M. Verhamme:
ovulatory follicles and cultured in the presence of lactate, 45 had formed a polar body at the first inspection. Thirty-one of these had degenerated but were still recognizable. In 7 oocytes a degenerated as well as an intact polar body was present. By regular inspection it could be seen that 24-31 hours after the onset of culture 22 new polar bodies appeared in the 31 oocytes which at the first inspection contained a degenerated polar body. In total therefore 29 out of 38 oocytes which had formed a polar body abstricted a second one later (Figs. l-3). Serial sections showed that both the degenerated and the second polar body contained chromatin. Of 74 denuded oocytes obtained from prepuberal ovaries and cultured in the presence of lactate, 37 had formed a polar body (19 of which had degenerated) at the first inspection. Three of these oocytes contained a degenerated as well as an intact polar body. Between 21 and 28 hours after incubation another 18 oocytes with a degenerated polar body extruded a second polar body.
Second polar body formed in rat ovarian oocytes
generated first polar body rules out the possibility that the first polar body had divided. It must be remarked that in those cases in which one intact polar body is found at first inspection about 20 hours after incubation, there is no certainty about its status (first or second) unless another polar body is extruded later. This uncertainty exists with regard to the oocytes cultured without added lactate. Contrary to the synchronous appearance of the first polar body (Zeilmaker and Verhamme, 1974) the second polar body appeared at an unpredictable time between 21 and 31 hours of incubation and in several cases also earlier. In tubal oocytes spontaneous formation of the second polar body does not occur. Apparently the in vitro conditions favor the extrusion.of this body not only in tubal oocytes (Zeilmaker and Verhamme, 1974) but also in oocytes matured in vitro.
References Austin, C.R. and Braden, A.W.H. (1954): Induction and inhibition of the second polar division in the rat egg and subsequent fertilization. Aust. J. biol. Sci., ,7, 195-210. Biggers, J.D., Whittingham, D.G. and Donahue, R.P. (1967): The pattern of energy metabolism in the mouse oocyte and zygote. Proc. nat. Acad. Sci. (Wash.), 58, 560-567. Odor, D.L. (1955): The temporal relationship of the first maturation division of rat ova to the onset of heat. Amer. J. Anat., 97,461-469.
Discussion
The observations mentioned above indicate that in rat oocytes a second polar body may be formed spontaneously about 24 hours after incubation, i.e. about 16 hours after extrusion of the first polar body. The fact that polar body formation was observed by regular inspection in the presence of an already de-
Thibault, C. (1949): L’oeuf des mammiferes, son dbeloppement parth&rog&tique. Ann. Sci, nat. Zool. Biol. anim., II, 133-219.
ZeiImaker, G.H. and Verhamme, C.M.P.M. (1974): Observations on rat oocyte maturation in vitro: morphology and energy requirements. Biol. Reprod., II,’ 145-152. Zeilmaker, G.H., Vermeiden, J.P.W., Verhamme, C.M.P.M. and Van Vliet, A.C.W. (1974): Observations on rat and mouse oocyte maturation in vivo and in vitro: morphology and physiology. Europ. J. Obstet. Gynec. reprod. Biol., 4, 15-24.
35-38. EXCERPTA MEDICA
Second polar body formation in rat ovarian oocytes matured in vitro G.H. Zeilmaker Department
and C.M.P.M.
of Endocrinology,
Verhamme
Growth and Reproduction,
Erasmus University, Rotterdam,
The Netherlands
ZEILMAKER, G.H. and VERHAMME, C.M.P.M. (1976): Second polar body formation in rat ovarian oocytes matured in vitro. Europ. J. Obstet. Gynec. reprod. Biol., 6/l,
35-38.
Denuded ovarian oocytes obtained from proestrous and prepuberal rats were incubated in medium containing lactate or without added energy source. Formation of a second polar body was observed about 24 h after the onset of culture. Formation by division of the fist polar body was excluded by observing second polar body formation in the presence of a degenerated first polar body. energy requirements; lactate; polar body degeneration
Introduction
Materials
and methods
Denuded rat ovarian oocytes will mature in vitro in media containing pyruvate or lactate and even in the absence of an added energy source. The first polar body is extruded 8-9 hours after explantation and often degenerates within several hours (Zeilmaker and Verhamme, 1974). It is well known that in the rat ovary the first polar body also degenerates soon after its formation even before follicle rupture (Odor, 1955; Zeilmaker, Vermeiden, Verhamme and Van Vliet, 1974). Formation of the second polar body in tubal oocytes can be induced by a variety of stimuli (Thibault, 1949; Austin and Braden, 1954) or by explantation of tubal oocytes in vitro (Zeilmaker and Verhamme, 1974). In the present study it was observed that in oocyte cultures which were regularly inspected, polar bodies were formed long after the expected time of first polar body formation. Data are presented which indicate that a second polar body may be formed in vitro in ovarian oocytes obtained from proestrous and prepuberal rats.
Ovarian oocytes were obtained from preovulatory follicles, dissected from 19 rats in proestrous (11 a.m., before expected LH surge) or by puncturing ovaries from twelve 30-day-old rats (3 p.m.). Cumulus cells were removed mechanically by pipetting and the washed oocytes obtained from one rat and containing a germinal vesicle were incubated under oil in a balanced salt solution (Biggers, Whittingham and Donahue, 1967) with 10 mM lactate or without added energy source. The cultures were gassed individually with 5% COz, 5% O2 and 90% N2 in a temperaturecontrolled room.
Results
Oocyte cultures were inspected for the formation of the first polar body 20-22 hours after explantation. At hourly intervals thereafter the number of oocytes with polar bodies was recorded. It appeared that of 62 oocytes isolated from pre35
36
G.H. Z&maker
and C.M.P.M. Verhamme:
Second polar body formed in rat ovarian oocytes
Fig. 1. Rat oocyte photographed twice during extrusion of a second polar body 24 h after explantation. During the interval between photographs (less than 5 mm) the oocyte turned clockwise about 30 degrees. Particles visible outside the zona pellucida near the degenerated polar body (top) are out of focus in the bottom photograph, and visible near the degenerated polar body through the zona.
G.H. Zeilmaker and C.M.P.M. Verhamme:
Second polar body formed in rat ovarian oocytes
Fig. 2. Rat oocyte with degenerated first polar body and a second polar body which has been fformed less than 2 h before photography.
Fig. 3. Rat oocyte with degenerated first polar body and fully formed second polar body.
31
38
G.H. Zeilmaker and C.M.P.M. Verhamme:
ovulatory follicles and cultured in the presence of lactate, 45 had formed a polar body at the first inspection. Thirty-one of these had degenerated but were still recognizable. In 7 oocytes a degenerated as well as an intact polar body was present. By regular inspection it could be seen that 24-31 hours after the onset of culture 22 new polar bodies appeared in the 31 oocytes which at the first inspection contained a degenerated polar body. In total therefore 29 out of 38 oocytes which had formed a polar body abstricted a second one later (Figs. l-3). Serial sections showed that both the degenerated and the second polar body contained chromatin. Of 74 denuded oocytes obtained from prepuberal ovaries and cultured in the presence of lactate, 37 had formed a polar body (19 of which had degenerated) at the first inspection. Three of these oocytes contained a degenerated as well as an intact polar body. Between 21 and 28 hours after incubation another 18 oocytes with a degenerated polar body extruded a second polar body.
Second polar body formed in rat ovarian oocytes
generated first polar body rules out the possibility that the first polar body had divided. It must be remarked that in those cases in which one intact polar body is found at first inspection about 20 hours after incubation, there is no certainty about its status (first or second) unless another polar body is extruded later. This uncertainty exists with regard to the oocytes cultured without added lactate. Contrary to the synchronous appearance of the first polar body (Zeilmaker and Verhamme, 1974) the second polar body appeared at an unpredictable time between 21 and 31 hours of incubation and in several cases also earlier. In tubal oocytes spontaneous formation of the second polar body does not occur. Apparently the in vitro conditions favor the extrusion.of this body not only in tubal oocytes (Zeilmaker and Verhamme, 1974) but also in oocytes matured in vitro.
References Austin, C.R. and Braden, A.W.H. (1954): Induction and inhibition of the second polar division in the rat egg and subsequent fertilization. Aust. J. biol. Sci., ,7, 195-210. Biggers, J.D., Whittingham, D.G. and Donahue, R.P. (1967): The pattern of energy metabolism in the mouse oocyte and zygote. Proc. nat. Acad. Sci. (Wash.), 58, 560-567. Odor, D.L. (1955): The temporal relationship of the first maturation division of rat ova to the onset of heat. Amer. J. Anat., 97,461-469.
Discussion
The observations mentioned above indicate that in rat oocytes a second polar body may be formed spontaneously about 24 hours after incubation, i.e. about 16 hours after extrusion of the first polar body. The fact that polar body formation was observed by regular inspection in the presence of an already de-
Thibault, C. (1949): L’oeuf des mammiferes, son dbeloppement parth&rog&tique. Ann. Sci, nat. Zool. Biol. anim., II, 133-219.
ZeiImaker, G.H. and Verhamme, C.M.P.M. (1974): Observations on rat oocyte maturation in vitro: morphology and energy requirements. Biol. Reprod., II,’ 145-152. Zeilmaker, G.H., Vermeiden, J.P.W., Verhamme, C.M.P.M. and Van Vliet, A.C.W. (1974): Observations on rat and mouse oocyte maturation in vivo and in vitro: morphology and physiology. Europ. J. Obstet. Gynec. reprod. Biol., 4, 15-24.