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Luteotropic influence of the ovine uterus
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 8(3):369-374,1991
LUTEOTROPIC INFLUENCE OF THE OVINE UTERUS A.P. Sankot and W.J. Murdoch Department of Animal Science University of Wyoming, Laramie WY 82071 Received September 14, 1990
ABSTRACT Preliminary observations indicated that the ovine uterus might play a contributing role in the development of the corpus luteum. In order to better define this putative relationship, we monitored hiteal function in mature ewes that were hysterectomized or sham-operated at different intervals following induction of ovulation. Corpora lutea formed following hysterectomy carried out immediately after ovulation were subnormal. Circulatory concentrations of progesterone in these animals began to ascend normally, but then achieved a plateau level less than that of control animals. This was attributed to reduced size of the luteal gland, and not to anomalies per unit tissue in morphology or content of progesterone. Luteal activity was not altered in ewes hysterectomized later in the estrous cycle (Day 5). However, when such a luteal phase was terminated by exogenous luteolysin, corpora lutea formed subsequently were defective. It appears that the ovine uterus produces a hormonal factor during metestrus that augments the growth potential of the corpus luteum.
INTRODUCTION The uterus has a pivotal role in the regulation of luteal function in sheep. The vast majority of research effort on this topic has centered around the participation of this organ in the mechanism of luteolysis (1). That the undecidualized uterus can exert a luteotropic effect is not widely recognized. Ovine corpora lutea formed following hysterectomy shortly after ovulation apparently begin to develop normally, but then function at an attenuated capacity (2). A similar phenomenon was reported when anestrous ewes were hysterectomized before induction of ovulation (3). Circulatory levels of progesterone are likewise suppressed following hysterectomy in horses (4, 5) and rabbits (6). Thus, it appears that in certain species the uterus produces a luteotropin. The objectives of the following experiments were to establish the critical period for manifestation of the uterine luteotropic influence in sheep and to determine the apparent nature of the insufficiency with regard to luteal function and morphology. MATERIALS AND METHODS Experiment 1. Western-range ewes were observed daily for estrous behavior in the presence of vasectomized rams. Fourteen d following detection of estms animals were injected intramuscularly with a luteolytic dose of prostaglandin (PG) F2a (10 mg dinoprost tromethamine; The Upjohn Co., Kalamazoo, MI) to synchronize the timing of luteal regression. A preovulatory surge in secretion of luteinizing hormone (LH) was induced 36 hr later by intramuscular injection of an agonistic analog of LH-releasing hormone (10 pg desGlyl°-AI#-LHRH ethylamide; LHRH; Sigma Chemical Co., St. Louis, MO). Ovulation occurs about 24 hr after administration of LHRH and the resulting luteal phase is typical of an unmanipulated estrous cycle (7). The day of administration of LHRH was considered Day 0 of the treatment cycle. Eight ewes per group were assigned at random to be either hysterectomized or sham-operated on Day 1. General anesthesia was induced and maintained by sodium thiopental Copyright © 1991 Butterworth-Heinemann
369
0739-7240/91/$3.00
370
SANKOT AND MURDOCH
given intravenously. Reproductive organs were exteriorized through a midventral abdominal incision using aseptic technique. Bilateral hysterectomy was carried out after placing ligatures around uterine/tubal vessels and the utero-cervical junction. Care was taken not to disturb blood vessels serving the ovaries. Sham-operated control animals were laparotomized and the reproductive tract handled before wound closure. Blood samples were obtained daily by jugular venipuncture from Days 2 through 12. Progesterone was measured in ether-extracted serum by validated radioimmunoassay (2). Differences in patterns of secretion of progesterone were compared by split-plot analysis of variance. Individual means within time were contrasted by Student's t-test. E x p e r i m e n t 2. The above experiment was repeated except that ewes were bysterectomized or sham-operated on Day 5 (eight animals/group) and serum samples for progesterone assay were obtained each day from Day 6 through 14. Experiment 3. Ewes (six/group) were hysterectomized or sham-operated on Day 5. Luteal regression was provoked by injection of PGF2a on Day 14. Blood samples for serum analyses of progesterone were collected daily beginning on Day 6 of the treatment cycle and ending on Day 12 of the ensuing luteal phase. Experiment 4. Corpora lutea were dissected on Day l0 from ovaries of animals (six/ group) that had been either hysterectomized or sham-operated on Day 1. Glands were cleaned of extraneous tissue, weighed and hemisected. One portion of tissue was immediately frozen in liquid nitrogen and stored at -70 C awaiting progesterone analysis (2). The other segment of luteal tissue was fixed by immersion in chilled 10% buffered formalin. Fixed tissues were processed for paraffin embedding/sectioning (6 microns) and hematoxylin and eosin staining using standard technique. Morphometric analyses were carried out on a minimum of ten fields selected at random per corpus luteum. Criteria of interest included areas of a particular tissue section represented by large luteal cells, small luteal cells and blood vessels. These determinations were performed with the aid of a microcomputer image-analysis system (8). An image of a tissue section was projected at 500 X onto a sensor pad coupled to a data analyzer. Perimeters of luteal and endothelial cells were traced and the cumulative areas occupied by cellular/vascular spaces computed as a percentage of total area represented. Large luteal cells were identified as spherical in shape with a diameter of approximately 20 microns or greater. Small cells had a lesser diameter and were stellate in shape. If more than one corpus luteum was obtained from an individual, average data values for that animal were calculated. Differences in treatment means were compared by Student's t-test. RESULTS Experiment 1. Luteal malfunction was exhibited following hysterectomy on Day 1. Serum concentrations of progesterone began to rise normally in hysterectomized ewes, but beyond Day 7 after administration of LHRH, were significantly lower than in sham-operated controls (Fig. 1). Experiment 2. There was no effect of hysterectomy v e r s u s sham-operation carried out on Day 5 on serum concentrations of progesterone throughout the ensuing nine d (Fig. 2). Experiment 3. When animals were hysterectomized on Day 5, and then luteal regression induced on Day 14, only the second luteal phase following surgical treatments was altered (ie., when ovulation and formation of the corpus luteum occurred in the absence of the uterus). The circulatory profile of progesterone during the aberrant luteal phase was similar to that encountered in Experiment 1 (Fig. 3). Experiment 4. Corpora lutea collected on Day 10 from ewes hysterectomized on Day 1 were lighter than glands isolated from control animals. However, there were no significant differences due to treatment in luteal concentrations of progesterone (Fig. 4) or histological parameters measured (Fig. 5).
UTERINE LUTEOTROPIN
371
PROGESTERONE (ng/ml) 3 •
HYSTERECTOMY
•
SHAM-OPERATION
__ _ _ . . ~ , ~
2
1
0 2
3
4
5
6
7
8
9
10
11
12
DAY AFTER LHRH Fig. 1. Serum concen~afions of p~gesterune in ewes h y s ~ t o m i z e d ~ sham-operated on DW 1. M e ~ s within day differed signific~tly (P < 0.05) f ~ m Day 8 through 12. Pool~ s ~ d error = 0.20.
PROGESTERONE (ng/ml) 3 •
HYSTERECTOMY
n SHAM-OPERATION 2
0 6
7
8
9
10
11
12
13
14
DAY AFTER LHRH Fig. 2. Serum concentrations of progesterone in ewes hysterectomized or sham-operated on Day 5. Pooled standard error = 0.18.
DISCUSSION It appears that in the sheep the uterus is involved not only in the ultimate demise of the corpus luteum, but somewhat paradoxically, has a luteotropic influence during its developmental stage. It is doubtful that overall output of progesterone by the corpus luteum following hysterectomy on Day 1 was curtailed as a consequence of surgical trauma. Luteal production of progesterone was not acutely affected. Nor did hysterectomy on Day 5 modify serum progesterone throughout the surgically-manipulated cycle. Rather, luteal insufficiency was only manifested when corpora lutea then formed in the absence of the uterus.
372
SANKOT AND MURDOCH
PROGESTERONE (ng/ml)
3 • HYSTERECTOMY
6
7
8
9
10 11 12 13 14 15 16 0
1
2
3
4
5
6
7
8
9
10 11 12
DAY AFTER LHRH Fig. 3. Serum concentrations of progesterone in ewes hysterectomized or sham-operated on Day 5. Means within day differed significantly (P < 0.05) from Day 7 through 12 of the second luteal phase post-surgery. Pooled standard error = 0.21.
70 •
L U T E A L WEIGHT (mg X 1 0 )
[]
PROGESTERONE
•
PROGESTERONE ( m i c r o g r a m s )
60 (ng/mg)
50 40 30 20 10 0 HYSTERECTOMY
SHAM-OPERATION
Fig. 4. Weights of corpora lutea and concentrations and contents of progesterone in luteal tissue of hysterectomized and shamoperated ewes. An asterisk indicates a difference (P < 0.05) between treatment means.
On a per unit mass basis, there was neither a functional or anatomical distinction between glands formed in the presence or absence of the uterus. Corpora lutea were simply smaller in the case of hysterectomy, and therefore, the overall capacity for ovarian secretion of progesterone was diminished. Furthermore, we have observed no differences due to hysterectomy in responsiveness of luteal tissue to LH in vitro (unpublished observation). A similar situation was confronted when subnormal ovine corpora lutea were examined following induction of ovulation during the anestrous season (9). The chemical identity of the purported uterine luteotropin, or whether this factor gains access to the ovary by a local or systemic vascular route, is unknown. Milvae and Hansel (I0) reported that bovine corpora lutea do not develop normally when the uterus is infused
UTERINE LUTEOTROPIN
373
% AREA 30
20
10
0 SHAM-OPERATION
HYSTERECTOMY [ ] LARGE CELLS
[ ] SMALL CELLS
[ ] BLOOD VESSELS Fig. 5. Areasof tissue sectionscomposedof large luteal cells, small luteal cells and bloodvessels.
with indomethacin and suggested that a cyclooxygenase derivative of arachidonate (most likely prostacyclin; 11) of ovarian or uterine origin was required for proper formation of the corpus luteum. The nonpregnant uterus of the human menstrual cycle produces prolactin (12), a hormone that possesses luteotropic properties in sheep (13). Intriguingly, prolactin was luteotropic in hypophysectomized, hysterectomized ewes (13), but not in hypophysectomized, uterine-intact animals (14). ACKNOWLEDGEMENTS/FOOTNOTES The technical assistance of E.A. Van Kirk is appreciated. Address requests for reprints and correspondence to WJM.
REFERENCES 1. Knickerbocker JJ, Wiltbank MC, Niswender GD. Mechanisms of luteolysis in domestic livestock. Domest Anim Endocrinol 5:91-107, 1988. 2. Rahrnanian MS, Murdoch WJ. Function of ovine corpora lutea after administration of luteinizing hormonereleasing hormone. J Anim Sci 64:648-655, 1987. 3. Southee JA, Hunter MG, Law AS, Haresign W. Effect of hysterectomy on the short life-cycle corpus luteum produced after GnRH-induced ovulation in the anoestrous ewe. J Reprod Fertil 84:149-155, 1988. 4. Stabenfeldt GH, Hughes JP, Wheat JD, Evans JW, Kennedy PC, Cupps PT. The role of the uterus in ovarian control in the mare. J Reprod Fertil 37:343-351, 1974. 5. Squires EL, Wentworth BC, Ointher OJ. Progesterone concentration in blood of mares during the estrous cycle, pregnancy and after hysterectomy. J Anim Sci 39:759-767, 1975. 6. Miller JB, Keyes PL. A mechanism for regression of the rabbit corpus luteum: uterine induced loss of luteal responsiveness to 1713-estradiol. Biol Reprod 15:511-518, 1976. 7. Roberts AJ, Duma TG, Murdoch WJ. Induction of ovulation in proestrous ewes: identification of the ovulatory follicle and functional status of the corpus luteum. Domest Anim Endocrinol 2:207-210, 1985. 8. Cavender JL, Murdoch WJ. Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 39:989-997, 1988. 9. O'Shea JD, Rodgers RJ, Wright PJ. Morphometric analysis and function in vivo and in vitro of corpora lutea from ewes treated with LHRH during seasonal anoestrus. J Reprod Fertil 72:75--85, 1984.
374
SANKOT AND MURDOCH
10. Milvae RA, Hansel W. Inhibition of bovine luteal function by indomethacin. J Anim Sci 60:528-531, 1985. 11. Milvae RA. Role of luteal prostaglandins in the control of bovine corpus luteum functions. J Anim Sci [Suppl 2] 62:72-78, 1986. 12. Monnier JC, Motte-Pouyol H. Ovarian function after hysterectomy. LARC Med Lille 4:405-410, 1984. 13. Denamur R, Martinet J, Short RV. Pituitary control of the ovine corpus luteum. J Reprod Fertil 32:207-220, 1973. 14. Kaltenbach CC, Graber JW, Niswender GD, Nalbandov AV. Luteotrophic properties of some pituitary hormones in nonpregnant or pregnant hypophysectomized ewes. Endocrinology 82:818-824, 1968.
Vol. 8(3):369-374,1991
LUTEOTROPIC INFLUENCE OF THE OVINE UTERUS A.P. Sankot and W.J. Murdoch Department of Animal Science University of Wyoming, Laramie WY 82071 Received September 14, 1990
ABSTRACT Preliminary observations indicated that the ovine uterus might play a contributing role in the development of the corpus luteum. In order to better define this putative relationship, we monitored hiteal function in mature ewes that were hysterectomized or sham-operated at different intervals following induction of ovulation. Corpora lutea formed following hysterectomy carried out immediately after ovulation were subnormal. Circulatory concentrations of progesterone in these animals began to ascend normally, but then achieved a plateau level less than that of control animals. This was attributed to reduced size of the luteal gland, and not to anomalies per unit tissue in morphology or content of progesterone. Luteal activity was not altered in ewes hysterectomized later in the estrous cycle (Day 5). However, when such a luteal phase was terminated by exogenous luteolysin, corpora lutea formed subsequently were defective. It appears that the ovine uterus produces a hormonal factor during metestrus that augments the growth potential of the corpus luteum.
INTRODUCTION The uterus has a pivotal role in the regulation of luteal function in sheep. The vast majority of research effort on this topic has centered around the participation of this organ in the mechanism of luteolysis (1). That the undecidualized uterus can exert a luteotropic effect is not widely recognized. Ovine corpora lutea formed following hysterectomy shortly after ovulation apparently begin to develop normally, but then function at an attenuated capacity (2). A similar phenomenon was reported when anestrous ewes were hysterectomized before induction of ovulation (3). Circulatory levels of progesterone are likewise suppressed following hysterectomy in horses (4, 5) and rabbits (6). Thus, it appears that in certain species the uterus produces a luteotropin. The objectives of the following experiments were to establish the critical period for manifestation of the uterine luteotropic influence in sheep and to determine the apparent nature of the insufficiency with regard to luteal function and morphology. MATERIALS AND METHODS Experiment 1. Western-range ewes were observed daily for estrous behavior in the presence of vasectomized rams. Fourteen d following detection of estms animals were injected intramuscularly with a luteolytic dose of prostaglandin (PG) F2a (10 mg dinoprost tromethamine; The Upjohn Co., Kalamazoo, MI) to synchronize the timing of luteal regression. A preovulatory surge in secretion of luteinizing hormone (LH) was induced 36 hr later by intramuscular injection of an agonistic analog of LH-releasing hormone (10 pg desGlyl°-AI#-LHRH ethylamide; LHRH; Sigma Chemical Co., St. Louis, MO). Ovulation occurs about 24 hr after administration of LHRH and the resulting luteal phase is typical of an unmanipulated estrous cycle (7). The day of administration of LHRH was considered Day 0 of the treatment cycle. Eight ewes per group were assigned at random to be either hysterectomized or sham-operated on Day 1. General anesthesia was induced and maintained by sodium thiopental Copyright © 1991 Butterworth-Heinemann
369
0739-7240/91/$3.00
370
SANKOT AND MURDOCH
given intravenously. Reproductive organs were exteriorized through a midventral abdominal incision using aseptic technique. Bilateral hysterectomy was carried out after placing ligatures around uterine/tubal vessels and the utero-cervical junction. Care was taken not to disturb blood vessels serving the ovaries. Sham-operated control animals were laparotomized and the reproductive tract handled before wound closure. Blood samples were obtained daily by jugular venipuncture from Days 2 through 12. Progesterone was measured in ether-extracted serum by validated radioimmunoassay (2). Differences in patterns of secretion of progesterone were compared by split-plot analysis of variance. Individual means within time were contrasted by Student's t-test. E x p e r i m e n t 2. The above experiment was repeated except that ewes were bysterectomized or sham-operated on Day 5 (eight animals/group) and serum samples for progesterone assay were obtained each day from Day 6 through 14. Experiment 3. Ewes (six/group) were hysterectomized or sham-operated on Day 5. Luteal regression was provoked by injection of PGF2a on Day 14. Blood samples for serum analyses of progesterone were collected daily beginning on Day 6 of the treatment cycle and ending on Day 12 of the ensuing luteal phase. Experiment 4. Corpora lutea were dissected on Day l0 from ovaries of animals (six/ group) that had been either hysterectomized or sham-operated on Day 1. Glands were cleaned of extraneous tissue, weighed and hemisected. One portion of tissue was immediately frozen in liquid nitrogen and stored at -70 C awaiting progesterone analysis (2). The other segment of luteal tissue was fixed by immersion in chilled 10% buffered formalin. Fixed tissues were processed for paraffin embedding/sectioning (6 microns) and hematoxylin and eosin staining using standard technique. Morphometric analyses were carried out on a minimum of ten fields selected at random per corpus luteum. Criteria of interest included areas of a particular tissue section represented by large luteal cells, small luteal cells and blood vessels. These determinations were performed with the aid of a microcomputer image-analysis system (8). An image of a tissue section was projected at 500 X onto a sensor pad coupled to a data analyzer. Perimeters of luteal and endothelial cells were traced and the cumulative areas occupied by cellular/vascular spaces computed as a percentage of total area represented. Large luteal cells were identified as spherical in shape with a diameter of approximately 20 microns or greater. Small cells had a lesser diameter and were stellate in shape. If more than one corpus luteum was obtained from an individual, average data values for that animal were calculated. Differences in treatment means were compared by Student's t-test. RESULTS Experiment 1. Luteal malfunction was exhibited following hysterectomy on Day 1. Serum concentrations of progesterone began to rise normally in hysterectomized ewes, but beyond Day 7 after administration of LHRH, were significantly lower than in sham-operated controls (Fig. 1). Experiment 2. There was no effect of hysterectomy v e r s u s sham-operation carried out on Day 5 on serum concentrations of progesterone throughout the ensuing nine d (Fig. 2). Experiment 3. When animals were hysterectomized on Day 5, and then luteal regression induced on Day 14, only the second luteal phase following surgical treatments was altered (ie., when ovulation and formation of the corpus luteum occurred in the absence of the uterus). The circulatory profile of progesterone during the aberrant luteal phase was similar to that encountered in Experiment 1 (Fig. 3). Experiment 4. Corpora lutea collected on Day 10 from ewes hysterectomized on Day 1 were lighter than glands isolated from control animals. However, there were no significant differences due to treatment in luteal concentrations of progesterone (Fig. 4) or histological parameters measured (Fig. 5).
UTERINE LUTEOTROPIN
371
PROGESTERONE (ng/ml) 3 •
HYSTERECTOMY
•
SHAM-OPERATION
__ _ _ . . ~ , ~
2
1
0 2
3
4
5
6
7
8
9
10
11
12
DAY AFTER LHRH Fig. 1. Serum concen~afions of p~gesterune in ewes h y s ~ t o m i z e d ~ sham-operated on DW 1. M e ~ s within day differed signific~tly (P < 0.05) f ~ m Day 8 through 12. Pool~ s ~ d error = 0.20.
PROGESTERONE (ng/ml) 3 •
HYSTERECTOMY
n SHAM-OPERATION 2
0 6
7
8
9
10
11
12
13
14
DAY AFTER LHRH Fig. 2. Serum concentrations of progesterone in ewes hysterectomized or sham-operated on Day 5. Pooled standard error = 0.18.
DISCUSSION It appears that in the sheep the uterus is involved not only in the ultimate demise of the corpus luteum, but somewhat paradoxically, has a luteotropic influence during its developmental stage. It is doubtful that overall output of progesterone by the corpus luteum following hysterectomy on Day 1 was curtailed as a consequence of surgical trauma. Luteal production of progesterone was not acutely affected. Nor did hysterectomy on Day 5 modify serum progesterone throughout the surgically-manipulated cycle. Rather, luteal insufficiency was only manifested when corpora lutea then formed in the absence of the uterus.
372
SANKOT AND MURDOCH
PROGESTERONE (ng/ml)
3 • HYSTERECTOMY
6
7
8
9
10 11 12 13 14 15 16 0
1
2
3
4
5
6
7
8
9
10 11 12
DAY AFTER LHRH Fig. 3. Serum concentrations of progesterone in ewes hysterectomized or sham-operated on Day 5. Means within day differed significantly (P < 0.05) from Day 7 through 12 of the second luteal phase post-surgery. Pooled standard error = 0.21.
70 •
L U T E A L WEIGHT (mg X 1 0 )
[]
PROGESTERONE
•
PROGESTERONE ( m i c r o g r a m s )
60 (ng/mg)
50 40 30 20 10 0 HYSTERECTOMY
SHAM-OPERATION
Fig. 4. Weights of corpora lutea and concentrations and contents of progesterone in luteal tissue of hysterectomized and shamoperated ewes. An asterisk indicates a difference (P < 0.05) between treatment means.
On a per unit mass basis, there was neither a functional or anatomical distinction between glands formed in the presence or absence of the uterus. Corpora lutea were simply smaller in the case of hysterectomy, and therefore, the overall capacity for ovarian secretion of progesterone was diminished. Furthermore, we have observed no differences due to hysterectomy in responsiveness of luteal tissue to LH in vitro (unpublished observation). A similar situation was confronted when subnormal ovine corpora lutea were examined following induction of ovulation during the anestrous season (9). The chemical identity of the purported uterine luteotropin, or whether this factor gains access to the ovary by a local or systemic vascular route, is unknown. Milvae and Hansel (I0) reported that bovine corpora lutea do not develop normally when the uterus is infused
UTERINE LUTEOTROPIN
373
% AREA 30
20
10
0 SHAM-OPERATION
HYSTERECTOMY [ ] LARGE CELLS
[ ] SMALL CELLS
[ ] BLOOD VESSELS Fig. 5. Areasof tissue sectionscomposedof large luteal cells, small luteal cells and bloodvessels.
with indomethacin and suggested that a cyclooxygenase derivative of arachidonate (most likely prostacyclin; 11) of ovarian or uterine origin was required for proper formation of the corpus luteum. The nonpregnant uterus of the human menstrual cycle produces prolactin (12), a hormone that possesses luteotropic properties in sheep (13). Intriguingly, prolactin was luteotropic in hypophysectomized, hysterectomized ewes (13), but not in hypophysectomized, uterine-intact animals (14). ACKNOWLEDGEMENTS/FOOTNOTES The technical assistance of E.A. Van Kirk is appreciated. Address requests for reprints and correspondence to WJM.
REFERENCES 1. Knickerbocker JJ, Wiltbank MC, Niswender GD. Mechanisms of luteolysis in domestic livestock. Domest Anim Endocrinol 5:91-107, 1988. 2. Rahrnanian MS, Murdoch WJ. Function of ovine corpora lutea after administration of luteinizing hormonereleasing hormone. J Anim Sci 64:648-655, 1987. 3. Southee JA, Hunter MG, Law AS, Haresign W. Effect of hysterectomy on the short life-cycle corpus luteum produced after GnRH-induced ovulation in the anoestrous ewe. J Reprod Fertil 84:149-155, 1988. 4. Stabenfeldt GH, Hughes JP, Wheat JD, Evans JW, Kennedy PC, Cupps PT. The role of the uterus in ovarian control in the mare. J Reprod Fertil 37:343-351, 1974. 5. Squires EL, Wentworth BC, Ointher OJ. Progesterone concentration in blood of mares during the estrous cycle, pregnancy and after hysterectomy. J Anim Sci 39:759-767, 1975. 6. Miller JB, Keyes PL. A mechanism for regression of the rabbit corpus luteum: uterine induced loss of luteal responsiveness to 1713-estradiol. Biol Reprod 15:511-518, 1976. 7. Roberts AJ, Duma TG, Murdoch WJ. Induction of ovulation in proestrous ewes: identification of the ovulatory follicle and functional status of the corpus luteum. Domest Anim Endocrinol 2:207-210, 1985. 8. Cavender JL, Murdoch WJ. Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 39:989-997, 1988. 9. O'Shea JD, Rodgers RJ, Wright PJ. Morphometric analysis and function in vivo and in vitro of corpora lutea from ewes treated with LHRH during seasonal anoestrus. J Reprod Fertil 72:75--85, 1984.
374
SANKOT AND MURDOCH
10. Milvae RA, Hansel W. Inhibition of bovine luteal function by indomethacin. J Anim Sci 60:528-531, 1985. 11. Milvae RA. Role of luteal prostaglandins in the control of bovine corpus luteum functions. J Anim Sci [Suppl 2] 62:72-78, 1986. 12. Monnier JC, Motte-Pouyol H. Ovarian function after hysterectomy. LARC Med Lille 4:405-410, 1984. 13. Denamur R, Martinet J, Short RV. Pituitary control of the ovine corpus luteum. J Reprod Fertil 32:207-220, 1973. 14. Kaltenbach CC, Graber JW, Niswender GD, Nalbandov AV. Luteotrophic properties of some pituitary hormones in nonpregnant or pregnant hypophysectomized ewes. Endocrinology 82:818-824, 1968.