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Variation in the oxytocin content of caprine corpora lutea across the breeding season
THERIOGENOLOCY
VARIATION IN THE OXYTOCIN CONTENT OF CAPNINE CORPCRA LUTEA ACROSS THE BREEDIN SEASON L. C. Freeman and W. 6. Currie Department of Animal Science Cornell University Ithaca, NY 14853 Received for publication: July 16, 1984 Accepted: November 29, 1984 ABSTBACT Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season. Key words: goat, corpora lutea, oxytocin, season INTRODUCTION The normal estrouscycle of the dairy goat lasts approximately 20 to 21 days. Abnormally short estrous cycles of less than 12 days, and often of only five to seven days, have been observed in seasonally breeding goats at the beginning of the breeding season (1,2). These abnormally short cycles have been associated with premature luteal failure (3). Administering exogenous oxytocin on days 3 to 6 (estrus = 0) significantly reduces the interestrus interval by interfering with luteal development (4,5). The caprine CL contains oxytocin (6). This objective of this study was to investigate the possibility that luteal oxytocin is a factor in short estrous cycles at the initiation of the breeding season. HATERIALS
AND METHODS
Nine goats from one to three years old were used. The goats were loosely housed under natural day length and temperature, on a diet of grass hay supplemented with concentrate. All goats were checked for estrus at
____________________ The authors wish to thank Frank Michel who assisted Acknowledgements: with the surgeries.
MARCH
1985
VOL. 23 NO. 3
481
THERIOGENOLOGY least twice daily and were considered to be estrous when mounting by a fertile buck fitted with an apron.
they stood for
Estrous activity was first evident in late September of 1983 when the study was initiated. Goats were allocated randomly into three groups (n=3). Corpora lutea were collected on day 6 of the estrous cycle at the beginning of the breeding season (October 3-lo), the middle of the breeding season (December 2-5) or the end of the breeding season (February 5-7). The goats, kept in individual pens, were fasted for 24 hours prior to surgery. Ovaries bearing corpora lutea were collected via mid-ventral laparotomy after anesthetizing with xylazine (RompunR, Bayvet, Shawnee, KS) and ketamine (KetasetR, Bristol Laboratories, Syracuse, NY). Corpora lutea were rapidly dissected from nonluteal ovarian tissue. A central slice of luteal tissue was processed for electron microscopy. The remainder of the CL was immediately frozen in liquid nitrogen, then stored at -200C. Peptides were extracted from the CL using a method based on that of Walsh and Niall (7). Two or three 100-mg aliquots were obtained from the CL while still semifrozen and homogenized (Polytron PT-10 Brinkman) for one to two minutes in ice cold medium to minimize proteolysis. The extraction medium consisted of 15% (v/v) trifluoroacetic acid, 5% (v/v) formic acid, 1% (v/v) sodium chloride and 1M HCl (4 ml medium/100 mg tissue). The homogenate was centrifuged at 28,000xg at 4oC for 20 minutes and the resulting pellet was re-extracted using the same procedure. The supernatants were filtered through a 0.45~ Millipore filter, then applied to a disposable octadecylsilica cartridge (Sep-Pak, Waters Associates Milford, MA). The Sep-Pak had been equilibrated with lOm1 of 80% (v/v) acetonitrile, 20% (v/v) aqueous trifluoroacetic acid (O.l%,v/v), followed by lOm1 distilled water. After the supernatants from both extractions had been pumped through the Sep-Pak, the effluents were combined and passed through a second time. The cartridges were washed with lOm1 of 10% (v/v) acetonitrile, 90% (v/v) aqueous trifluoroacetic acid (O.l%,v/v); the effluent was discarded. Ovarian peptides were eluted using l.Oml of 80% acetonitrile, 20% aqueous trifluoroacetic acid solution. The eluted fraction was volumetrically diluted at least lo-fold in radioimmunoassay buffer, pH 7.5 (8) and further dilutions, as appropriate, were prepared prior to RIA. Residual solvent at the dilutions used did not interfere with measuring immunoreactive oxytocin. Oxytocin concentrations were determined in samples by an RIA procedure modified from that of Gorewit (8,9). Oxytocin antiserum was obtained from Calbiochem-Behring (LaJolla, CA). Lyophilized aliquots of antisera (lot 142108) were diluted 1:150 using normal rabbit serum (1:400) prior to use in the assay. The final dilution of antiserum per assay tube was 1:lOOO. Synthetic oxytocin (Calbiochem-Behring, LaJolla, CA) was used as standard in all assays. The peptide is lyophilized in the presence of mannitol as an excipient which results in a mass potency of 23.2 IU/mg of powder. All measurements were converted to I U using this potency value. Synthetic oxytocin was radioiodinated using a chloramine T reaction. 50ug oxytocin, 1 mCi, Na1251 and 55ug of chloramine T were combined in the presence of 0.5M phosphate buffer (pH 7.5); after 25 seconds, the reaction was quenched by adding lmg L-tyrosine methyl ester hydrochloride (Sigma, St. Louis, MO). A column (0.9 x 50cm) packed with Bio-Gel P-2 (200-400 mesh, Bio Rad LaboraMARCH
1985 VOL. 23 NO. 3
THERIOGENOLOGY
tories, Richmond, CA) was used to separate iodinated oxytocin from iodide. The gel was prewashed with 1Oml of 10% BSA in 0.5M PBS followed by 50ml of the elution buffer (0.05M sodium phosphate, pH 7.5, 0.002% sodium azide). Data were evaluated by analysis of variance. Differences between means were compared using least significant differences. RESULTS AND DISCUSSION The oxytocin concentrations in samples of ovarian tissue collected at different stages of the breeding season are shown in Table 1. The variability within and between animals was high. Despite the considerable variability within groups at each stage, the concentrations of oxytocin in luteal tissue collected during the early breeding season were significantly greater than those in luteal tissue collected during the mid and late breeding season (Table 2). The considerable variation in oxytocin content of portions of the CL may be the result of nonuniform distribution. The ruminant CL contains distinct populations of cells including small and large luteal cells, endothelial cells, fibroblasts, and myoid cells. Data from sheep indicate that only large luteal cells contain oxytocin (10). As lUO-mg aliquots of luteal tissue are unlikely to contain all cell types in similar proportions, such aliquots are also unlikely to contain equal concentrations of oxytocin. In a preliminary experiment, oxytocin concentrations were determined in five 100-mg aliquots obtained from one entire CL; the coefficient of variation was 75%. Caprine luteal tissue contained more oxytocin than nonluteal ovarian tissue (Table 1). This finding is consistent with measurements of oxytocin in ovarian tissues of other species (11-13). The oxytocin content variation in aliquots of nonluteal ovarian tissue may be related to the distribution of follicles. In the cow, the concentrations of oxytocin in the fluid of large follicles were intermediate between the concentrations in the CL and the concentrations in other ovarian tissue (12). Administering oxytocin early in the caprine estrous cycle consistently inhibits luteal development and shortens the interestrus interval (4, Freeman and Currie, unpublished). Increasing evidence suggests that this luteolytic action of oxytocin is not a purely pharmacologic phenomenon. The corpora lutea of domestic ruminants including the goat contain high concentrations of oxytocin (6,11,12) and luteal oxytocin has been implicated (14) in normal luteal regression.
MARCH
1985 VOL. 23 NO. 3
483
Table 1. Oxytocin concentrations in ovarian tissue from goats at early, mid and late stages of the breeding season. Non Luteala (IU/g wet weight)
Luteal (IU/g wet weight) EARLY 2.39, 3.80, 7.28b
0.11, 0.02
1.77,
3.94,
0.02,
3.64,
0.62
0.67
0.02
0.10
Mean + SEM 3.01 + 0.77 __~_~__~__~_~__~_~_---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ MID 0.27, 0.52
0.02, 0.02
0.85, 0.31
0.02, 0.00
0.17,
0.04
0.22
0.39 t 0.11 _____~~_____~_____~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Mean t SEM LATE
Mean + SEM
0.63, 1.03
0.01, 0.01
0.59, 2.39
0.04, 0.00
0.70,
0.01,
0.43
0.01
0.96 2 0.30
a Portions of ovary remaining after enucleation of the corpus luteum; there was no systematic selection for or against follicle size. b Values on the same line are from replicate samples.
484
tissue aliquots
within
MARCH 1985VOL. 23 NO. 3
THERIOCENOLOGY
Table 2.
Analysis of variance of luteal concentrations of oxytocin
Source
d.f.
Total
19
F
M.S.
Stages
2
13.60
aResidual
17
2.13
6.38
(P
L.S.D.
[
Early
Late
Mid
3.01
0.96
0.39
impcO.05
+
NS-
1
a Pooled within stages, i.e. within and between animals The data presented here show that oxytocin is present in caprine corpora lutea in significantly higher concentrations during the early breeding season than during the mid or late breeding season. The high concentrations of luteal oxvtocin early in the breeding season may be responsible for the high incidence of premature luteal regression and -abnormally short estrous cycles at this time. REFERENCES R.S. Some aspects of caprine theriogenology. Proc. Ann. Meeting Sot. for Theriogenology, Spokane, Washington, 1981, pp. 152-159.
1.
Ott,
2.
Riera, S. Reproductive efficiency and management in goats. Proc. Int. Conf. Goat Prod. Disease, Tucson, Arizona, 1982, pp. 162174.
3.
Camp, J.C., Wildt, D.E., Howard, P.K., Stuart, L. and Chakraborty, P.K. Ovarian activity during normal and abnormal length of estrous cycles in the goat. Biol. Reprod -28:673-681 (1983).
4.
Cooke, R.G. and Knifton, A. Oxytocin-induced Theriogenology -16:95-97 (1981).
5.
Cooke, R.G. and Homeida, A.M. Plasma concentrations of 13, 14 dihydro-15"keto-prostaglandin F2o and progesterone during oxytocininduced oestrus in the goat. Theriogenology -18:453-460 (1982).
MARCH
1985 VOL. 23 NO. 3
oestrus
in the goat.
485
THERIOGENOLOCY
6.
Homeida, A.M. and Cooke, K.G. Evidence that oxytocin is of luteal origin during the caprine oestrus cycle. Proc. Sot. for Study of Fertility, London, England, 1983, p. 15 (Abstr.)
7.
Walsh, J.R. and Niall, H.D. Use of an octadecylsilica purification method minimizes proteolysis during isolation of porcine and rat relaxins. Endocrinology -107:1258-1260 (1980).
8.
Gorewit, R.C. Method for determining oxytocin concentrations in unextracted sera; characterization in lactating cattle. Proc. Sot. Exp. Biol. Med. 160:80-87 (1979).
9.
Wachs, E.A., Gorewit, R.C. and Currie, W.B. Half-life clearance and production rate for oxytocin in cattle during lactation and mammary involution. Dam. Anim. Endo. 1:121-40 (1984).
10. Rodgers, R.J., O'Shea, J.D., Findlay, J.K., Flint, A.P. and Sheldrick, E.L. Large luteal cells the source of oxytocin in the sheep. Endocrinology -113:2302-2304 (1983). 11.
Wathes, D.C. and Swann, R.W. 297:225-227 (1982).
Is oxytocin an ovarian hormone?
Nature
12. Schams, D., Walters, D.L., Schallenberger, E., Bullerman, B. and Karg, H. Ovarian oxytocin in the cow. Acta Endocrinol. Suppl. 253:147. (Abstr., 1983). 13. Khan-Dawood, F.S. and Dawood, M.Y. Human ovaries contain immunoreactive oxytocin. J. Clin. Endocrinol. Metab. -57:1129-1132 (1983). 14. Flint, A.P.F. and Sheldrick, E.L. Evidence for a systemic role for J. Reprod. Fertil. ovarian oxytocin in luteal regression in sheep. (1983). -67:215-255
486
MARCH
1985 VOL. 23 NO. 3
VARIATION IN THE OXYTOCIN CONTENT OF CAPNINE CORPCRA LUTEA ACROSS THE BREEDIN SEASON L. C. Freeman and W. 6. Currie Department of Animal Science Cornell University Ithaca, NY 14853 Received for publication: July 16, 1984 Accepted: November 29, 1984 ABSTBACT Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season. Key words: goat, corpora lutea, oxytocin, season INTRODUCTION The normal estrouscycle of the dairy goat lasts approximately 20 to 21 days. Abnormally short estrous cycles of less than 12 days, and often of only five to seven days, have been observed in seasonally breeding goats at the beginning of the breeding season (1,2). These abnormally short cycles have been associated with premature luteal failure (3). Administering exogenous oxytocin on days 3 to 6 (estrus = 0) significantly reduces the interestrus interval by interfering with luteal development (4,5). The caprine CL contains oxytocin (6). This objective of this study was to investigate the possibility that luteal oxytocin is a factor in short estrous cycles at the initiation of the breeding season. HATERIALS
AND METHODS
Nine goats from one to three years old were used. The goats were loosely housed under natural day length and temperature, on a diet of grass hay supplemented with concentrate. All goats were checked for estrus at
____________________ The authors wish to thank Frank Michel who assisted Acknowledgements: with the surgeries.
MARCH
1985
VOL. 23 NO. 3
481
THERIOGENOLOGY least twice daily and were considered to be estrous when mounting by a fertile buck fitted with an apron.
they stood for
Estrous activity was first evident in late September of 1983 when the study was initiated. Goats were allocated randomly into three groups (n=3). Corpora lutea were collected on day 6 of the estrous cycle at the beginning of the breeding season (October 3-lo), the middle of the breeding season (December 2-5) or the end of the breeding season (February 5-7). The goats, kept in individual pens, were fasted for 24 hours prior to surgery. Ovaries bearing corpora lutea were collected via mid-ventral laparotomy after anesthetizing with xylazine (RompunR, Bayvet, Shawnee, KS) and ketamine (KetasetR, Bristol Laboratories, Syracuse, NY). Corpora lutea were rapidly dissected from nonluteal ovarian tissue. A central slice of luteal tissue was processed for electron microscopy. The remainder of the CL was immediately frozen in liquid nitrogen, then stored at -200C. Peptides were extracted from the CL using a method based on that of Walsh and Niall (7). Two or three 100-mg aliquots were obtained from the CL while still semifrozen and homogenized (Polytron PT-10 Brinkman) for one to two minutes in ice cold medium to minimize proteolysis. The extraction medium consisted of 15% (v/v) trifluoroacetic acid, 5% (v/v) formic acid, 1% (v/v) sodium chloride and 1M HCl (4 ml medium/100 mg tissue). The homogenate was centrifuged at 28,000xg at 4oC for 20 minutes and the resulting pellet was re-extracted using the same procedure. The supernatants were filtered through a 0.45~ Millipore filter, then applied to a disposable octadecylsilica cartridge (Sep-Pak, Waters Associates Milford, MA). The Sep-Pak had been equilibrated with lOm1 of 80% (v/v) acetonitrile, 20% (v/v) aqueous trifluoroacetic acid (O.l%,v/v), followed by lOm1 distilled water. After the supernatants from both extractions had been pumped through the Sep-Pak, the effluents were combined and passed through a second time. The cartridges were washed with lOm1 of 10% (v/v) acetonitrile, 90% (v/v) aqueous trifluoroacetic acid (O.l%,v/v); the effluent was discarded. Ovarian peptides were eluted using l.Oml of 80% acetonitrile, 20% aqueous trifluoroacetic acid solution. The eluted fraction was volumetrically diluted at least lo-fold in radioimmunoassay buffer, pH 7.5 (8) and further dilutions, as appropriate, were prepared prior to RIA. Residual solvent at the dilutions used did not interfere with measuring immunoreactive oxytocin. Oxytocin concentrations were determined in samples by an RIA procedure modified from that of Gorewit (8,9). Oxytocin antiserum was obtained from Calbiochem-Behring (LaJolla, CA). Lyophilized aliquots of antisera (lot 142108) were diluted 1:150 using normal rabbit serum (1:400) prior to use in the assay. The final dilution of antiserum per assay tube was 1:lOOO. Synthetic oxytocin (Calbiochem-Behring, LaJolla, CA) was used as standard in all assays. The peptide is lyophilized in the presence of mannitol as an excipient which results in a mass potency of 23.2 IU/mg of powder. All measurements were converted to I U using this potency value. Synthetic oxytocin was radioiodinated using a chloramine T reaction. 50ug oxytocin, 1 mCi, Na1251 and 55ug of chloramine T were combined in the presence of 0.5M phosphate buffer (pH 7.5); after 25 seconds, the reaction was quenched by adding lmg L-tyrosine methyl ester hydrochloride (Sigma, St. Louis, MO). A column (0.9 x 50cm) packed with Bio-Gel P-2 (200-400 mesh, Bio Rad LaboraMARCH
1985 VOL. 23 NO. 3
THERIOGENOLOGY
tories, Richmond, CA) was used to separate iodinated oxytocin from iodide. The gel was prewashed with 1Oml of 10% BSA in 0.5M PBS followed by 50ml of the elution buffer (0.05M sodium phosphate, pH 7.5, 0.002% sodium azide). Data were evaluated by analysis of variance. Differences between means were compared using least significant differences. RESULTS AND DISCUSSION The oxytocin concentrations in samples of ovarian tissue collected at different stages of the breeding season are shown in Table 1. The variability within and between animals was high. Despite the considerable variability within groups at each stage, the concentrations of oxytocin in luteal tissue collected during the early breeding season were significantly greater than those in luteal tissue collected during the mid and late breeding season (Table 2). The considerable variation in oxytocin content of portions of the CL may be the result of nonuniform distribution. The ruminant CL contains distinct populations of cells including small and large luteal cells, endothelial cells, fibroblasts, and myoid cells. Data from sheep indicate that only large luteal cells contain oxytocin (10). As lUO-mg aliquots of luteal tissue are unlikely to contain all cell types in similar proportions, such aliquots are also unlikely to contain equal concentrations of oxytocin. In a preliminary experiment, oxytocin concentrations were determined in five 100-mg aliquots obtained from one entire CL; the coefficient of variation was 75%. Caprine luteal tissue contained more oxytocin than nonluteal ovarian tissue (Table 1). This finding is consistent with measurements of oxytocin in ovarian tissues of other species (11-13). The oxytocin content variation in aliquots of nonluteal ovarian tissue may be related to the distribution of follicles. In the cow, the concentrations of oxytocin in the fluid of large follicles were intermediate between the concentrations in the CL and the concentrations in other ovarian tissue (12). Administering oxytocin early in the caprine estrous cycle consistently inhibits luteal development and shortens the interestrus interval (4, Freeman and Currie, unpublished). Increasing evidence suggests that this luteolytic action of oxytocin is not a purely pharmacologic phenomenon. The corpora lutea of domestic ruminants including the goat contain high concentrations of oxytocin (6,11,12) and luteal oxytocin has been implicated (14) in normal luteal regression.
MARCH
1985 VOL. 23 NO. 3
483
Table 1. Oxytocin concentrations in ovarian tissue from goats at early, mid and late stages of the breeding season. Non Luteala (IU/g wet weight)
Luteal (IU/g wet weight) EARLY 2.39, 3.80, 7.28b
0.11, 0.02
1.77,
3.94,
0.02,
3.64,
0.62
0.67
0.02
0.10
Mean + SEM 3.01 + 0.77 __~_~__~__~_~__~_~_---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ MID 0.27, 0.52
0.02, 0.02
0.85, 0.31
0.02, 0.00
0.17,
0.04
0.22
0.39 t 0.11 _____~~_____~_____~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Mean t SEM LATE
Mean + SEM
0.63, 1.03
0.01, 0.01
0.59, 2.39
0.04, 0.00
0.70,
0.01,
0.43
0.01
0.96 2 0.30
a Portions of ovary remaining after enucleation of the corpus luteum; there was no systematic selection for or against follicle size. b Values on the same line are from replicate samples.
484
tissue aliquots
within
MARCH 1985VOL. 23 NO. 3
THERIOCENOLOGY
Table 2.
Analysis of variance of luteal concentrations of oxytocin
Source
d.f.
Total
19
F
M.S.
Stages
2
13.60
aResidual
17
2.13
6.38
(P
L.S.D.
[
Early
Late
Mid
3.01
0.96
0.39
impcO.05
+
NS-
1
a Pooled within stages, i.e. within and between animals The data presented here show that oxytocin is present in caprine corpora lutea in significantly higher concentrations during the early breeding season than during the mid or late breeding season. The high concentrations of luteal oxvtocin early in the breeding season may be responsible for the high incidence of premature luteal regression and -abnormally short estrous cycles at this time. REFERENCES R.S. Some aspects of caprine theriogenology. Proc. Ann. Meeting Sot. for Theriogenology, Spokane, Washington, 1981, pp. 152-159.
1.
Ott,
2.
Riera, S. Reproductive efficiency and management in goats. Proc. Int. Conf. Goat Prod. Disease, Tucson, Arizona, 1982, pp. 162174.
3.
Camp, J.C., Wildt, D.E., Howard, P.K., Stuart, L. and Chakraborty, P.K. Ovarian activity during normal and abnormal length of estrous cycles in the goat. Biol. Reprod -28:673-681 (1983).
4.
Cooke, R.G. and Knifton, A. Oxytocin-induced Theriogenology -16:95-97 (1981).
5.
Cooke, R.G. and Homeida, A.M. Plasma concentrations of 13, 14 dihydro-15"keto-prostaglandin F2o and progesterone during oxytocininduced oestrus in the goat. Theriogenology -18:453-460 (1982).
MARCH
1985 VOL. 23 NO. 3
oestrus
in the goat.
485
THERIOGENOLOCY
6.
Homeida, A.M. and Cooke, K.G. Evidence that oxytocin is of luteal origin during the caprine oestrus cycle. Proc. Sot. for Study of Fertility, London, England, 1983, p. 15 (Abstr.)
7.
Walsh, J.R. and Niall, H.D. Use of an octadecylsilica purification method minimizes proteolysis during isolation of porcine and rat relaxins. Endocrinology -107:1258-1260 (1980).
8.
Gorewit, R.C. Method for determining oxytocin concentrations in unextracted sera; characterization in lactating cattle. Proc. Sot. Exp. Biol. Med. 160:80-87 (1979).
9.
Wachs, E.A., Gorewit, R.C. and Currie, W.B. Half-life clearance and production rate for oxytocin in cattle during lactation and mammary involution. Dam. Anim. Endo. 1:121-40 (1984).
10. Rodgers, R.J., O'Shea, J.D., Findlay, J.K., Flint, A.P. and Sheldrick, E.L. Large luteal cells the source of oxytocin in the sheep. Endocrinology -113:2302-2304 (1983). 11.
Wathes, D.C. and Swann, R.W. 297:225-227 (1982).
Is oxytocin an ovarian hormone?
Nature
12. Schams, D., Walters, D.L., Schallenberger, E., Bullerman, B. and Karg, H. Ovarian oxytocin in the cow. Acta Endocrinol. Suppl. 253:147. (Abstr., 1983). 13. Khan-Dawood, F.S. and Dawood, M.Y. Human ovaries contain immunoreactive oxytocin. J. Clin. Endocrinol. Metab. -57:1129-1132 (1983). 14. Flint, A.P.F. and Sheldrick, E.L. Evidence for a systemic role for J. Reprod. Fertil. ovarian oxytocin in luteal regression in sheep. (1983). -67:215-255
486
MARCH
1985 VOL. 23 NO. 3