Elemental P. S.
composition of rabbit antral fluid during preovulatory follicular swelling
Burgoyne,
R. M.
Borland, J. D. Biggers and C. P. Lechene
Laboratory of Human Reproduction and Reproductive Biology, Department ofPhysiology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, U.SA.
Summary. Electron probe microanalysis was used to determine the concentrations of
Na, Cl, K, Ca, Mg, S and P in samples of follicular fluid, ovarian vein
serum and 2-h 10 h after obtained from rabbits intervals at to up virgin peripheral injection of hCG. Throughout this 10-h period the elemental composition of follicular fluid was essentially the same as that of blood serum. However, there was a significant drop in follicular fluid Ca relative to blood during the 10-h period which may reflect Ca involvement in the regulation of oocyte maturation. Significant differences were also found between follicles within rabbits for K and P concentrations. venous serum
Introduction Meiotic maturation in the rabbit resumes after the LH surge following coitus. Approximately 10 h later ovulation occurs with the release of an ovum in the metaphase II stage of meiosis (Blandau, 1971). These processes can be induced artificially by the injection of human chorionic gonadotrophin (hCG) (Harper, 1963). During this 10-h interval the Graafian follicle swells by about 60% (Rondell, 1964) due to the accumulation of solutes and water in the antrum. The elemental composition of the antral fluid in the preovulatory period has not been studied previously, and is the subject of the present report. Elucidation of this composition is important in understanding the mechanisms involved in follicular expansion and in describing the microenvironment in which the oocyte matures.
Materials and Methods
Animals
Post-pubertal virgin Dutch Belted rabbits were used. Each rabbit was caged individually for 3 weeks before use to avoid pseudopregnancy. Samples of peripheral blood, ovarian venous blood and follicular fluid were obtained from rabbits 2, 4, 6, 8 and 10 h after the injection of 25 i.u. hCG into the marginal ear vein. Samples were also obtained from uninjected rabbits which were assumed to be in oestrus (denoted 0 h). Three rabbits were allotted at random to each of the 6 treatment groups. Since no more than 2 rabbits could be sampled per day, this randomization procedure was necessary to ensure that the results were not influenced by secular trends in the responses of the rabbits or to technical trends during the course of the investigation. About 20 min before surgery each rabbit received 0-3 ml tribromoethanol in tertiary amyl Present address: Department of Obstetrics Edinburgh EH3 9EW, U.K. *
&
Gynaecology, University of Edinburgh, 37 Chalmers Street,
alcohol (1 g/ml, w/v)/kg body weight. The anaesthetic was given rectally in 10 ml warm water and within 3 min the rabbit was semiconscious. Sufficient sodium pentobarbitone was given, 5 min before surgery, through the ear vein to quell the reflex in the Achilles tendon. This procedure allowed exact control of the time of surgery, and largely avoided the depressive effects of sodium pentobarbitone on the circulation.
Sample collection and preparation Follicular fluid was collected from the anaesthetized rabbits using siliconized hand-held pipettes 30-80 pm in tip diameter. These pipettes were drawn by hand from capillary tubing. A truncated Beem capsule was placed against the ovary to create a well over a Graafian follicle, and was filled with mineral oil which had been equilibrated with 0-9% (w/v) NaCl. Equilibrated mineral oil was used throughout the study. A pipette filled with mineral oil was inserted into the antrum of the follicle and a sample of fluid was withdrawn. More oil was drawn into the pipette, which was then sealed in a flame and centrifuged on an IEC microhaematocrit centrifuge to remove
any cells.
Samples of ovarian venous blood were also collected using the hand-held pipettes immediately after the sampling of the follicular fluids. The blood was taken up slowly to avoid haemolysis. After centrifugation to remove cells, the sample was allowed to clot, and the serum was collected after further centrifugation. Peripheral blood was collected from an ear vein before anaesthesia, either with hand-held pipettes or with a 1 ml syringe fitted with a 26-gauge needle. Again serum was collected after clotting and centrifugation. All three supernatant fluids were placed under mineral oil in a plastic Petri dish (Falcon No. 3001). Duplicate one-third dilutions of each fluid were subsequently prepared using a siliconized volumetric micropipette. When diluting the follicular fluid samples, it was often necessary to stir the sample to separate fluid from clotted material that was present. The extent of clotting was variable.
Electron probe microanalysis A detailed description of the techniques involved is given by Lechene (1974), Borland, Biggers & Lechene (1976), and by Lechene & Warner (1977). Briefly, microdroplets of the diluted samples together with a series of standard solutions were placed with calibrated volumetric pipettes (35-80 pi) onto the surface of a beryllium support covered with mineral oil. The pipettes were made on a Sensaur microforge de Fonbrune, and calibrated with tritiated H20. The pipettes were rinsed in boiling deionized water and resiliconized between sample droplets. In this manner, accurate delivery of the entire picolitre volume was ensured. The oil on the beryllium block was removed with an m-xylene wash. The sample microdroplets on the block were immediately frozen in isopentane at —150°C, and then freeze-dried under vacuum (0-013 Pa, —70°C) so that very small crystals (
composition of rabbit antral fluid during preovulatory follicular swelling
Burgoyne,
R. M.
Borland, J. D. Biggers and C. P. Lechene
Laboratory of Human Reproduction and Reproductive Biology, Department ofPhysiology, Harvard Medical School, 45 Shattuck Street, Boston, Massachusetts 02115, U.SA.
Summary. Electron probe microanalysis was used to determine the concentrations of
Na, Cl, K, Ca, Mg, S and P in samples of follicular fluid, ovarian vein
serum and 2-h 10 h after obtained from rabbits intervals at to up virgin peripheral injection of hCG. Throughout this 10-h period the elemental composition of follicular fluid was essentially the same as that of blood serum. However, there was a significant drop in follicular fluid Ca relative to blood during the 10-h period which may reflect Ca involvement in the regulation of oocyte maturation. Significant differences were also found between follicles within rabbits for K and P concentrations. venous serum
Introduction Meiotic maturation in the rabbit resumes after the LH surge following coitus. Approximately 10 h later ovulation occurs with the release of an ovum in the metaphase II stage of meiosis (Blandau, 1971). These processes can be induced artificially by the injection of human chorionic gonadotrophin (hCG) (Harper, 1963). During this 10-h interval the Graafian follicle swells by about 60% (Rondell, 1964) due to the accumulation of solutes and water in the antrum. The elemental composition of the antral fluid in the preovulatory period has not been studied previously, and is the subject of the present report. Elucidation of this composition is important in understanding the mechanisms involved in follicular expansion and in describing the microenvironment in which the oocyte matures.
Materials and Methods
Animals
Post-pubertal virgin Dutch Belted rabbits were used. Each rabbit was caged individually for 3 weeks before use to avoid pseudopregnancy. Samples of peripheral blood, ovarian venous blood and follicular fluid were obtained from rabbits 2, 4, 6, 8 and 10 h after the injection of 25 i.u. hCG into the marginal ear vein. Samples were also obtained from uninjected rabbits which were assumed to be in oestrus (denoted 0 h). Three rabbits were allotted at random to each of the 6 treatment groups. Since no more than 2 rabbits could be sampled per day, this randomization procedure was necessary to ensure that the results were not influenced by secular trends in the responses of the rabbits or to technical trends during the course of the investigation. About 20 min before surgery each rabbit received 0-3 ml tribromoethanol in tertiary amyl Present address: Department of Obstetrics Edinburgh EH3 9EW, U.K. *
&
Gynaecology, University of Edinburgh, 37 Chalmers Street,
alcohol (1 g/ml, w/v)/kg body weight. The anaesthetic was given rectally in 10 ml warm water and within 3 min the rabbit was semiconscious. Sufficient sodium pentobarbitone was given, 5 min before surgery, through the ear vein to quell the reflex in the Achilles tendon. This procedure allowed exact control of the time of surgery, and largely avoided the depressive effects of sodium pentobarbitone on the circulation.
Sample collection and preparation Follicular fluid was collected from the anaesthetized rabbits using siliconized hand-held pipettes 30-80 pm in tip diameter. These pipettes were drawn by hand from capillary tubing. A truncated Beem capsule was placed against the ovary to create a well over a Graafian follicle, and was filled with mineral oil which had been equilibrated with 0-9% (w/v) NaCl. Equilibrated mineral oil was used throughout the study. A pipette filled with mineral oil was inserted into the antrum of the follicle and a sample of fluid was withdrawn. More oil was drawn into the pipette, which was then sealed in a flame and centrifuged on an IEC microhaematocrit centrifuge to remove
any cells.
Samples of ovarian venous blood were also collected using the hand-held pipettes immediately after the sampling of the follicular fluids. The blood was taken up slowly to avoid haemolysis. After centrifugation to remove cells, the sample was allowed to clot, and the serum was collected after further centrifugation. Peripheral blood was collected from an ear vein before anaesthesia, either with hand-held pipettes or with a 1 ml syringe fitted with a 26-gauge needle. Again serum was collected after clotting and centrifugation. All three supernatant fluids were placed under mineral oil in a plastic Petri dish (Falcon No. 3001). Duplicate one-third dilutions of each fluid were subsequently prepared using a siliconized volumetric micropipette. When diluting the follicular fluid samples, it was often necessary to stir the sample to separate fluid from clotted material that was present. The extent of clotting was variable.
Electron probe microanalysis A detailed description of the techniques involved is given by Lechene (1974), Borland, Biggers & Lechene (1976), and by Lechene & Warner (1977). Briefly, microdroplets of the diluted samples together with a series of standard solutions were placed with calibrated volumetric pipettes (35-80 pi) onto the surface of a beryllium support covered with mineral oil. The pipettes were made on a Sensaur microforge de Fonbrune, and calibrated with tritiated H20. The pipettes were rinsed in boiling deionized water and resiliconized between sample droplets. In this manner, accurate delivery of the entire picolitre volume was ensured. The oil on the beryllium block was removed with an m-xylene wash. The sample microdroplets on the block were immediately frozen in isopentane at —150°C, and then freeze-dried under vacuum (0-013 Pa, —70°C) so that very small crystals (
