Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the distribution of sizes of ovine luteal cells during the estrous cycle

The ovine corpus luteum is composed of two types of steroidogenic cells, which are referred to as small and large luteal cells. In this study, the size and number of steroidogenic cells were determined in corpora lutea collected on Days 4, 8, 12, and 16 of the estrous cycle. Corpora lutea were dissociated into single-cell suspensions that were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, a marker for steroidogenic cells. The size of 3 beta-HSD-positive cells was measured with a Zeiss Videoplan Image Analyzer. On Day 4, most of the 3 beta-HSD-positive cells were less than 18 microns in diameter, the median being 11.2 microns. By Day 8, the number of 3 beta-HSD-positive cells increased 3-fold, and the median diameter increased to 12.8 microns. Although the number of 3 beta-HSD-positive cells was reduced by approximately 50% on Day 16, the median size on Days 12 and 16 was 14.6 and 16.8 microns, respectively. The ratio of large (greater than 18 microns) to small (less than 18 microns) luteal cells was 0.11 +/- 0.03 on Day 4; the ratio increased linearly to 0.67 +/- 0.09 by Day 16. This increase between Days 4 and 12 was attributable to an overall increase in the size of the cells; the increase between Days 12 and 16, however, was due to a loss of small luteal cells. When the experiment was conducted near the end of the breeding season, before animals became anestrous, the median size of the luteal cells did not change at different times of the estrous cycle but remained constant throughout. These data suggest that development of the corpus luteum is associated with an increase in the size and number of steroidogenic luteal cells, and that luteolysis is associated with a preferential loss of small luteal cells.     

Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.     

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

Cellular composition of the sheep corpus luteum in the mid- and late luteal phases of the oestrous cycle

Corpora lutea (CL) from naturally cycling Corriedale ewes were obtained in the mid- and late luteal phases of the oestrous cycle (Days 9 and 13; 5 ewes per group). The cellular composition of these CL was compared by ultrastructural morphometry to determine whether there were changes in numbers of large and small luteal cells consistent with differentiation of some small luteal cells to large luteal cells during the last part of the luteal phase. No differences between Days 9 and 13 were detected in luteal volume, plasma progesterone concentration, or volume density of any component of the luteal tissue. Large luteal cell numbers (mean +/- s.e.m.) were lower per unit volume of luteal tissue on Day 13 than on Day 9 (14.1 +/- 0.5 vs 18.4 +/- 1.3 X 10(3)/mm3, P less than 0.05). Mean volume of the individual large luteal cells was greater on Day 13 than on Day 9 (19.65 +/- 0.72 vs' 15.60 +/- 1.34 micrograms 3 X 10(3), P less than 0.05). However, there were no significant differences in numbers or volumes of small luteal cells between Days 9 and 13, and total numbers of large luteal cells per CL were not different between these two days. These results provide no support for the hypothesis that small luteal cells differentiate into large luteal cells during the oestrous cycle of the sheep.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

A morphometric analysis of the corpus luteum of the cow during the estrous cycle and early pregnancy

Corpora lutea were collected from cows on Days 6, 8, 10, 12, 14, 16, 18 and 19 of the estrous cycle and early pregnancy (n=2/d) and were examined by light microscopy. Mean lutein cell diameter was significantly (P<0.05) greater in pregnant than in cyclic cows on Days 6, 8, 10, 12, 16, 18 and 19 (cyclic versus pregnant: Day 6: 13.9 +/- 0.22 vs 14.9 +/- 0.24; Day 8: 13.8 +/- 0.20 vs 15.4 +/- 0.2; Day 10: 14.8 +/- 0.24 vs 17.4 +/- 0.24; Day 12: 13.2 +/-0.25 vs 17.9 +/- 0.31; Day 16: 13.9 +/- 0.28 vs 16.5 +/- 0.31; Day 18: 13.0 +/- 0.22 vs 16.5 +/- 09.36, and Day 19: 15.0 +/- 0.23 vs 17.6 +/- 0.33 mum, respectively). The distribution of cell sizes was leptokurtotic throughout the estrous cycle and the first 10 d of pregnancy, but tended towards bimodality after Day 14 of pregnancy. The proportion of lutein cell cytoplasm occupied by vacuoles was lower in pregnant than in cyclic cows from the 12th day post estrus, but there was a marked (P<0.05) increase in vacuolation of cells from cows undergoing luteolysis. Stainable intercellular collagen was also less abundant in pregnant than cyclic cows from the 12th day post estrus. The higher rate of progesterone secretion of pregnant, compared with cyclic cows may be attributed to the greater numbers and greater contribution to luteal mass of large lutein cells in the corpus luteum of pregnancy.     

Quantification of receptors for estradiol-17 beta and progesterone in the pituitary and hypothalamus of gilts during the estrous cycle and early pregnancy

Nuclear and cytoplasmic exchange assays were utilized to quantify receptors for estradiol-17 beta (E2) and progesterone (P4) in hypothalamic and pituitary tissues from 4-6 gilts each on Days 1, 5, 10, 15 and 18 of the estrous cycle and from 4-5 gilts each on Days 5, 10, 15, 21 and 30 of pregnancy. No differences in the number of cytoplasmic E2 or P4 receptors in the pituitary were found from Days 1 to 15 of the estrous cycle (P greater than 0.05). However, on Day 18, the quantities of E2 and P4 receptors were 64-fold and 25-fold lower (P less than 0.01) than those found during Days 1 to 15 of the estrous cycle. No differences in the number of nuclear receptors for E2 in the pituitary were observed from Days 1 to 18 of the estrous cycle, but nuclear receptors for P4 were 2-fold higher (P less than 0.01) on Day 1 than Days 5 to 18. In hypothalamic tissue, the numbers of cytoplasmic and nuclear receptors for E2 and P4 were lower (P less than 0.05) on Day 18 than Day 10 of the cycle. The quantity of most steroid receptors decreased between Days 15 and 18 in nonpregnant gilts as luteolysis occurred and a new follicular phase was initiated. Pregnant pigs on Days 5, 10 and 15 had decreased pituitary receptors for E2 and P4 when compared with cycling animals on these days. In general, numbers of receptors in hypothalamic tissue did not differ between pregnant and nonpregnant pigs except for decreased (P less than 0.01) nuclear P4 receptors on Day 15.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.     

Weight, composition, mitosis, cell death and content of progesterone and DNA in the corpus luteum of pregnancy in the ewe

Changes in luteal weight from about Day 20 to near term, and in quantitative histology as assessed by ultrastructural morphometry and light microscopic counts of mitosis and cell death on Days 30, 60, 100 and 142, were studied in 168 pregnant ewes. Luteal weight (mean +/- s.d.) remained constant at 0.56 +/- 0.11 g until Day 120, and fell thereafter to reach 0.31 +/- 0.11 g after Day 140 (P less than 0.01). Up to Day 100, quantitative aspects of the composition of the luteal tissue showed no significant change, and values for volume density, cytoplasmic:nuclear ratio, cell number/mm3 and cell volume were comparable to values previously obtained for corpora lutea (CL) of the cycle. By Day 142 structural evidence of luteal regression was present, but regressive changes were much more marked in some CL than others. Mitosis was seen in a few cells (0.02-0.04%) on all of the days studied, but never in large luteal cells. Cell death was rarely seen up to Day 100, but had increased in incidence by Day 142 (P less than 0.01). Luteal progesterone content, 55.2 +/- 15.9 nmol/g on Day 30, was not significantly changed on Days 60, 100 or 142. It is concluded that (1) structural regression of the CL of pregnancy does not begin until much later than the time (about Day 50) when pregnancy ceases to depend on the CL; (2) structural luteal regression begins before parturition, but its time of onset and/or rate of progression vary widely between animals; and (3) large and small luteal cells remain as distinctive populations throughout pregnancy, and their numbers at all stages can be accounted for by survival of the cells which differentiate during the genesis of the CL.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Luteinizing hormone secretion and corpus luteum function in cows receiving two levels of progesterone

The objectives of this experiment were to determine if subnormal levels of progesterone (P4) indicative of luteal insufficiency influence (1) pulsatile release of luteinizing hormone (LH), (2) the interval to the preovulatory surge of LH after removal of P4, and (3) the secretion of P4 during the estrous cycle subsequent to administration of subnormal levels of P4. On Day 5 (Day = 0 day of estrus) of the estrous cycle, cows received P4-releasing intravaginal devices (PRID) to produce normal (2 PRIDs; n = 7) or subnormal (0.5 PRID; n = 6) concentrations of P4. Five cows served as controls. On Day 10, serial blood samples were collected from all cows. Collection of blood samples was again initiated on Day 17 in cows receiving PRIDs. The PRIDs were removed and blood collection continued for 78 h. Daily blood samples were collected from all animals for 42 days subsequent to estrus (estrous cycles 1 and 2, respectively). During estrous cycle 1, mean concentration of P4 was lower (p less than 0.05) and frequency of pulses of LH was higher (p less than 0.05) in cows receiving subnormal P4 than in cows receiving normal P4 and control cows. Plasma concentrations of estradiol (E2) were higher (p less than 0.05) on Days 9-16 of estrous cycle 1 in cows receiving subnormal P4 than in cows receiving normal P4 or in control cows. Concentrations of E2 were greater (p less than 0.05) at 6, 18, and 30 h following removal of PRIDs in cows receiving subnormal P4 than in cows receiving normal P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between variation in conceptus development and differences in estrous cycle duration in ewes

The objective of this study was to examine conceptus development on Day 13 in ewes with estrous cycles of different durations. Ewes (n = 80) were screened according to the length of their estrous cycles. Subsequently, ewes that had either SHORT or LONG cycles were utilized (15.9 +/- 0.1 or 18.6 +/- 0.4 days; mean +/- SEM, p less than 0.01; 10 ewes per group). Jugular blood samples were collected twice daily from Days 0-6 after mating and then once a day until slaughter on Day 13. Concentrations of progesterone in plasma and amounts of ovine trophoblast protein-1 (oTP-1), protein, and prostaglandins (PG) E2 and F2 alpha (PGF2 alpha) in uterine flushings were determined. Concentrations of progesterone were greater (Day by treatment interaction, p less than 0.01) on Days 2-4 for ewes in the SHORT group. On Day 5 and thereafter, progesterone concentrations were not different between groups. More (p less than 0.05) oTP-1 and protein (8.1 +/- 1.3 micrograms and 1.8 +/- 0.3 micrograms versus 2.4 +/- 1.3 micrograms and 0.8 +/- 0.3 mg) were recovered from uterine flushings from ewes in the SHORT versus LONG groups, respectively. The ratio of PGE2:PGF2 alpha was higher (p less than 0.06) in flushings from ewes in the SHORT versus LONG group (1.4 +/- 0.2 versus 0.9 +/- 0.2, respectively). Conceptuses were classified by stage of morphological development. Conceptus development was accelerated (p less than 0.01) in ewes of the SHORT group, as shown by filamentous conceptuses recovered from 78% versus 0% of SHORT versus LONG ewes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between insulin and glucose metabolism and pituitary-ovarian functions in fasted heifers

The effects of fasting between Days 8 and 16 of the estrous cycle on plasma concentrations of luteinizing hormone (LH), progesterone, cortisol, glucose and insulin were determined in 4 fasted and 4 control heifers during an estrous cycle of fasting and in the subsequent cycle after fasting. Cortisol levels were unaffected by fasting. Concentrations of insulin and glucose, however, were decreased (p less than 0.05) by 12 and 36 h, respectively, after fasting was begun and did not return to control values until 12 h (insulin) and 4 to 7 days (glucose) after fasting ended. Concentrations of progesterone were greater (p less than 0.05) in fasted than in control heifers from Day 10 to 15 of the estrous cycle during fasting, while LH levels were lower (p less than 0.01) in fasted than in control heifers during the last 24 h of fasting. Concentrations of LH increased (p less than 0.01) abruptly in fasted heifers in the first 4 h after they were refed on Day 16 of the fasted cycle. Concentrations (means +/- SEM) of LH also were greater (p less than 0.05) in fasted (11.2 +/- 2.6 ng/ml) than in control (4.7 +/- 1.2 ng/ml) heifers during estrus of the cycle after fasting; this elevated LH was preceded by a rebound response in insulin levels in the fasted-refed heifers, with insulin increasing from 176 +/- 35 pg/ml to 1302 +/- 280 pg/ml between refeeding and estrus of the cycle after fasting. Concentrations of LH, glucose and insulin were similar in both groups after Day 2 of the postfasting cycle. Concentrations of progesterone in two fasted heifers and controls were similar during the cycle after fasting, whereas concentrations in the other fasted heifers were less than 1 ng/ml until Day 10, indicating delayed ovulation and (or) reduced luteal function. Thus, aberrant pituitary and luteal functions in fasted heifers were associated with concurrent fasting-induced changes in insulin and glucose metabolism.     

Effect of epostane, a competitive inhibitor of the 3beta-hydroxysteroid dehydrogenase enzyme, in cyclic sows

The effect of epostane, a 3beta-hydroxysteroid dehydrogenase (3beta-HSD) inhibitor, on the levels of plasma cortisol and progesterone and on the length of the estrous cycle in sows was examined. Epostane was administered orally on Days 0 to 2 (n = 3), Days 4 to 6 (n = 3), Days 10 to 12 (n = 2) or Days 17 to 19 (n = 3) of the estrous cycle, or subcutaneously on Day 0 (n = 3), Day 4 (n = 3), Day 10 (n = 4) or Day 17 (n = 3). Eleven days after the first dose of epostane, the treatments were repeated. One group of sows (n = 3) that was bled during a single estrous cycle served as controls. Cortisol levels in each of the eight groups of sows that received epostane did not differ (P>0.05) from those in control sows. In contrast, progesterone was lowered (P<0.01) when epostane was given by injection on Day 4, 10 or 17, or when given orally on Days 4 to 6 and 10 to 12. Although epostane reduced progesterone levels, the estrous cycle was not shortened. The interestrous interval for the sows (n = 14) that completed their experimental estrous cycle before they were sacrificed at approximately one week after the last dose of epostane was 21.6 +/- 2.71 d. It was concluded that epostane, as administered in this study, lowered progesterone levels but did not shorten the estrous cycle.     

Prostaglandin F2 alpha-induced release of oxytocin from bovine corpora lutea in vitro

Two experiments were conducted to study the in vitro effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2), and luteinizing hormone (LH) on oxytocin (OT) release from bovine luteal tissue. Luteal concentration of OT at different stages of the estrous cycle was also determined. In Experiment 1, sixteen beef heifers were assigned randomly in equal numbers (N = 4) to be killed on Days 4, 8, 12, and 16 of the estrous cycle (Day 0 = day of estrus). Corpora lutea were collected, an aliquot of each was removed for determination of initial OT concentration, and the remainder was sliced and incubated with vehicle (control) or with PGF2 alpha (10 ng/ml), PGE2 (10 ng/ml), or LH (5 ng/ml). Luteal tissue from heifers on Day 4 was sufficient only for determination of initial OT levels. Luteal OT concentrations (ng/g) increased from 414 +/- 84 on Day 4 to 2019 +/- 330 on Day 8 and then declined to 589 +/- 101 on Day 12 and 81 +/- 5 on Day 16. Prostaglandin F2 alpha induced a significant in vitro release of luteal OT (ng.g-1.2h-1) on Day 8 (2257 +/- 167 vs. control 1702 +/- 126) but not on Days 12 or 16 of the cycle. Prostaglandin E2 and LH did not affect OT release at any stage of the cycle studied. In Experiment 2, six heifers were used to investigate the in vitro dose-response relationship of 10, 20, and 40 ng PGF2 alpha/ml of medium on OT release from Day 8 luteal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of human chorionic gonadotropin injections to sustain gonadotropin-releasing hormone-induced corpora lutea in postpartum beef cows

Anestrous postpartum (PP) Hereford cows (n =20) were used to determine the effects of repeated injections of human chorionic gonadotropin (hCG) on the progesterone (P4) secretion and functional lifespan of gonadotropin-releasing hormone (GnRH)-induced corpora lutea (CL). Suckling was reduced to once a day from Day 21 to Day 25 PP, and all cows received injections of 200 micrograms GnRH at 1500 h on Day 24 PP to induce ovulation. Treated cows (HCG, n = 10) received 200 IU hCG b.i.d. from 1900 h on Day 27 PP to 1900 h on Day 33 PP; control cows (CTRL, n=10) were not injected. Blood was collected on Days 21, 23, 25, and 27 to 33, 35, 37, and 39 PP. Serum P4 concentration was measured by radioimmunoassay and used to classify luteal lifespan and the associated estrous cycle as short (SHORT) or normal (NORM) in duration. Treatment with hCG resulted in more (p less than 0.01) cows with SHORT cycles (7 of 9 vs. 4 of 9). Serum P4 concentrations were similar (p greater than 0.20) between groups from 4 days before until 6 days after GnRH injection. Cows with NORM cycles (n = 7) had greater serum P4 concentrations (p less than 0.05) on Days 7 to 11 after GnRH than cows with SHORT cycles (n = 11). By Day 39 PP, all cows with SHORT cycles appeared to have undergone a second ovulation. Charcoal-stripped serum pools from before (PRE) and during hCG injection (INJ) were assayed for total luteinizing hormone-like bioactivity (LH-BA) using a dispersed mouse-Leydig cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)