Effects of luteinizing hormone on luteal cell populations in hypophysectomized ewes

To examine the effect of purified LH on development and function of luteal cells, 27 ewes were assigned to: (1) hypophysectomy plus 2 micrograms ovine LH given i.v. at 4-h intervals from Days 5 to 12 of the oestrous cycle (oestrus = Day 0; Group H + LH; N = 7); (2) hypophysectomy with no LH replacement (Group N-LH; N = 6); (3) control (no hypophysectomy) plus LH replacement as in Group H + LH (Group S + LH; N = 7); (4) control with no LH treatment (Group S-LH; N = 7). Blood samples were collected at 4-h intervals throughout the experiment to monitor circulating concentrations of LH, cortisol and progesterone. On Day 12 of the oestrous cycle corpora lutea were collected and luteal progesterone concentrations, unoccupied receptors for LH and number and sizes of steroidogenic and non-steroidogenic luteal cell types were determined. Corpora lutea from ewes in Group H-LH were significantly smaller (P less than 0.05), had lower concentrations of progesterone, fewer LH receptors, fewer small luteal cells and fewer non-steroidogenic cells than did corpora lutea from ewes in Group S-LH. The number of large luteal cells was unaffected by hypophysectomy, but the sizes of large luteal cells, small luteal cells and fibroblasts were reduced. LH replacement in hypophysectomized ewes maintained luteal weight and the numbers of small steroidogenic and non-steroidogenic luteal cells at levels intermediate between those observed in ewes in Groups L-LH and S-LH. In Group H + LH ewes, luteal and serum concentrations of progesterone, numbers of luteal receptors for LH, and the sizes of all types of luteal cells were maintained. Numbers of small steroidogenic and non-steroidogenic cells were also increased by LH in hypophysectomized ewes. In Exp. II, 14 ewes were assigned to: (1) sham hypophysectomy with no LH replacement therapy (Group S-LH; N = 5); (2) sham hypophysectomy with 40 micrograms ovine LH given i.v. at 4-h intervals from Day 5 to Day 12 of the oestrous cycle (Group S + LH; N = 5); and (3) hypophysectomy plus LH replacement therapy (Group H + LH; N = 4). Experimental procedures were similar to those described for Exp. I. Treatment of hypophysectomized ewes with a larger dose of LH maintained luteal weight, serum and luteal progesterone concentrations and the numbers of steroidogenic and non-steroidogenic luteal cells at control levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe

Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 micrograms) with (+P) and without (-P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the -P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes. All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and -P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the -P animals only exhibited a decline in progesterone production in vitro on Day 4 (P less than 0.01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the -P animals also decreased in weight (P less than 0.01) and progesterone content (P less than 0.001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only. These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.     

Changes in lipid contents and fatty acid compositions in ovine corpora lutea during the estrous cycle and early pregnancy

Changes in lipid contents and fatty acid compositions of each lipid fraction were examined in corpora lutea from 34 unmated ewes between Days 8 and 16 of the estrous cycle and from 6 ewes at Day 16 of pregnancy. Four patterns were observed during advancement of the estrous cycle. Luteal concentrations of free cholesterol and triglyceride (neutral lipids) increased between Days 14 and 16, during luteal regression, in a manner approximated by exponential functions of time, whereas luteal concentrations of phospholipid (polar lipids) increased and then decreased between Days 8 and 16 in a manner approximated by a sin function of time. Likewise, within the various lipid class component fatty acids, changes in palmitic acid weight percentages were approximated by sin functions of time, whereas arachidonic acid weight percentages increased between Days 14 and 16 in a manner approximated by exponential functions of time. Pregnancy either inhibited or reversed the changes in luteal lipid profiles, especially arachidonic acid percentages, between Days 14 and 16 of the estrous cycle. Luteal lipid profiles of corpora lutea from Day 16 pregnant sheep approximated lipid profiles of corpora lutea recovered from sheep between Days 12 and 14 of the estrous cycle. Comparison of luteal lipid profiles after tissue incubations at either 0 or 37 degrees C for 2 h revealed an effect of reproductive status on fatty acid metabolisms at Day 16. Changes observed in luteal lipid contents and fatty acid compositions during advancement of the estrous cycle represent aspects of lutein cell maturation and impending senescence that can be inhibited or reversed by pregnancy.     

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.     

Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes

Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content. Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively. The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD). The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation. Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured. Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells. However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes. These differences appear to be an amplification of basic function. Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.     

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.     

Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2 alpha receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep

Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2 alpha (PGF-2 alpha) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P less than 0.05) to PGF-2 alpha treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P less than 0.05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17 beta, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P less than 0.10) to have fewer small luteal cells and fewer (P less than 0.05) low-affinity PGF-2 alpha binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2 alpha; (ii) this abnormal luteal tissue is functionally competent for 2-3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of small luteal cells without a significant change in luteal mass by Day 3 and, overall, have fewer low-affinity PGF-2 alpha binding sites.     

Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in LH-RH-treated anoestrous ewes.

Nineteen Scottish Blackface ewes were given LH-RH (3 X 30 micrograms i.v., 90-min intervals) during anoestrus when prolactin levels were elevated. Plasma levels of prolactin were suppressed with CB 154 (twice daily, i.m.) on Days -5 to 0 (N = 5), 0 to +5 (N = 5) or -5 to +5 (N = 5) around the day of LH-RH treatment (Day 0). Control animals (N = 4) received saline on Days -5 to +5. Nine animals ovulated forming corpora lutea as judged by laparoscopy on Day +7. No difference in FSH or LH levels was found between treatments and ovulations occurred equally in all treatment groups. Progesterone levels were less than ng/ml in all animals up to Day 14. It is concluded that short-term suppression of prolactin does not affect the incidence of ovulation or corpus luteum progesterone production in LH-RH-treated anoestrous ewes.     

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.     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

The role of luteal steroid hormones in the regulation of the estrous cycle of high fecundity Olkuska sheep

The study was undertaken to investigate the steroid hormone production by sheep luteal cells. Corpora lutea were collected from 30 Olkuska sheep on Days 3, 6, 9, 12 and 15 of the estrous cycle during the reproductive season. In Experiment 1, steroid hormone concentration was estimated in extracts of CL. In Experiment 2, luteal cells were cultured in vitro for 24 h. Luteal cells isolated on Days 9 and 12 secreted high amounts of progesterone and androgens but smaller amounts of estradiol. Concentration of these steroids in CL extracts collected on the same days showed the same trend. In CL harvested on Day 15, a decrease in androgens and progesterone as well as a significant increase in estradiol were observed in culture media and in extracts. Judging from the high amounts of estradiol and low amounts of androgen observed at the end of the luteal phase, we speculate that the steroid hormones secreted by the regressing CL may play an active role in the regulation of the estrous cycle in the Olkuska sheep with autocrine influence on the luteal activity or a possible paracrine action on follicular growth.In the third Experiment, the possibility of heterogeneity in the multiple corpora lutea population of prolific Olkuska sheep was investigated. Differences were found in the level of progesterone and estradiol secretion by individual corpora lutea recovered from the same animal, which also varied in terms of weight. This is the first study which shows the existence of intra-ovarian and individual heterogeneity between corpora lutea recovered from ewes during the normal estrous cycle.     

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.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.     

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.     

Immunocytochemical localization of neurophysin and oxytocin in ovine corpora lutea

The cellular distribution of neurophysin and oxytocin within ovine corpora lutea obtained on Days 4, 10 and 16 of the estrous cycle was examined immunocytochemically. Serial sections (8-10 micron-thick) prepared from corpora lutea that had been fixed in Bouin's solution and embedded in paraffin were immunostained for neurophysin or oxytocin using the peroxidase-antiperoxidase (PAP) procedure. Irrespective of the day of the cycle examined, immunoreactivity was restricted to large luteal cells. However, on Days 4 and 10 of the cycle, the intensity of staining in large luteal cells was highly variable; and, within the same section some cells were heavily stained, others were only lightly stained, and still others were not stained at all. In contrast, on Day 16 of the cycle, the intensity of staining was uniform and essentially all of the large luteal cells were immunoreactive. Based on the results obtained, it is evident that immunoreactive neurophysin and oxytocin can be detected as early as Day 4 of the cycle, persists through Day 15, and is restricted to large luteal cells.     

Effects of progestagen treatment on concentrations of prostaglandins and oxytocin in plasma from the posterior vena cava of post-partum beef cows

The role of PGF-2 alpha in determining the lifespan of corpora lutea in the post-partum beef cow was investigated. In control cows (N = 5) induced to ovulate at Day 28 to 36 post partum by injection of 1000 i.u. hCG, corpora lutea had an average lifespan of only 8 days. In cows pretreated with 6 mg implants of a progestagen (norgestomet, N = 4) for 9 days, with implant removal 2 days before injection of hCG, luteal lifespan averaged 17.5 days. Concentrations of PGF-2 alpha in 9 hourly samples of plasma collected from the posterior vena cava via indwelling catheters were higher on Days 4 through 9 after injection of hCG (P less than 0.05) in the cows with short-lived corpora lutea. Greater release of PGF-2 alpha could therefore be a major factor in premature luteal regression. Concentrations of PGFM and oxytocin did not differ between cows with corpora lutea of short or normal lifespan. In a second experiment, concentrations of PGF-2 alpha in plasma from the posterior vena cava were examined during treatment with norgestomet (N = 8) or in contemporary controls (N = 7). In progestagen-treated cows, PGF-2 alpha was higher than in control cows (P less than 0.05), beginning on Day 3 of treatment and peaking on Day 5. It is concluded that the post-partum uterus increases secretion of PGF-2 alpha very early after first exposure to endogenous or exogenous progestagen.     

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.     

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.     

Suppression of oxytocin-induced prostaglandin F2alpha release after intra-uterine nordihydroguariaretic acid administration in ewes

Intrauterine administration of the 5-lipoxygenase inhibitor nordihydroguariaretic acid (NDGA; 5 mg, bid) on Days 9-14 of the ovine estrous cycle (estrus = Day 0) delayed luteolysis and extended the duration of the estrous cycle (20+/-1, SD, vs. 16+/-1 days; P < 0.01). In control ewes, plasma concentrations of 13,14,dihydro-15-keto prostaglandin F2alpha increased significantly (P < 0.001) following i.v. administration of oxytocin (10 i.u.) on Day 14; in the nordihydroguariaretic acid-treated ewes, however, there was no such increase. In addition, concentrations of endometrial oxytocin receptors were significantly less (P < 0.01) in the nordihydroguariaretic acid-treated ewes (218+/-60 vs. 579+/-66 fmol/mg tissue). These results suggest that 5-lipoxygenase products of arachidonate metabolism may be involved in the control of ovine luteal function.