Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers

Ultrasonography was used once daily to quantify corpora lutea, central luteal cavities, and luteinized tissue during interovulatory intervals (n = 66) and during Days 0 to 60 of pregnancy (n = 14) in nulliparous Holstein heifers (ovulation = Day 0). The corpus luteum of the estrous cycle was detectable by ultrasonography in most heifers from the day of ovulation (mean, Day 0.5) and extending into the regressive phase beyond the next ovulation (mean, Day 1.4 +/-0.2 after the next ovulation). During pregnancy, the corpus luteum was detected until Day 60 (end of study). Maximal central luteal cavity area detected on Days 0 to 20 was used retrospectively to group luteal glands into four cavity categories: no, small, medium, and large. These categories corresponded to approximate cavity diameters of <2 mm, 2 to 5 mm, 6 to 10 mm, and >10 mm, respectively. The incidence of each cavity category was similar between interovulatory intervals and pregnancies (combined incidence, 17 80 , 8 80 , 33 80 , and 22 80 for no, small, medium, and large cavities, respectively; total with cavities, 63 80 , 79%). Mean day of first detection of a central cavity was earliest for large cavities during interovulatory intervals (means, Days 4.7, 4.4, and 3.0 for small, medium, and large cavities, respectively; P<0.04) and during pregnancies (means, Days 5.5, 4.2, and 3.3, respectively; NS). However, the day that the cavities reached maximum size (range of means, Days 5.5 to 7.0) did not differ among categories. Mean day of last detection of the central cavity was significantly different among cavity categories during interovulatory intervals (means, Days 9.3, 11.1, and 17.4 for small, medium, and large cavities, respectively) and pregnancies (means, Days 7.0, 8.8, and 20.2, respectively). Time of loss of central cavities was similar between nonbred and pregnant heifers, and there was no significant difference among cavity categories in the length of the interovulatory interval (mean, 20.1 d). Luteal tissue area was not significantly different among cavity categories during interovulatory intervals. There were no indications that cavities were functionally important. Luteal tissue area increased linearly in pregnant heifers on Days 21 to 60 (mean slope, 2.6 mm(2)/day).     

Characterization of plasma membrane lipids and luteinizing hormone receptors of ovine corpora lutea during luteolysis and early pregnancy

Lipid composition of plasma membranes from luteal cells was examined to determine whether changes in this organelle occur during regression and maintenance of the corpus luteum in nonpregnant (NP) and pregnant (P) ewes, respectively. Forty ewes were assigned to be killed on Day 13 or 15 of the estrous cycle (D13-NP and D15-NP) or pregnancy (D13-P and D15-P). Purification of luteal plasma membranes on discontinuous sucrose gradients yielded two fractions, designated F1 and F2, that exhibited the greatest enrichment of 5'-nucleotidase activity (five- and fourfold, respectively) over that of the homogenate. These fractions also yielded the lowest contamination by endoplasmic reticulum as represented by nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C reductase activity and mitochondrial membranes as indicated by succinate dehydrogenase activity. Predominant phospholipids identified in membranes obtained from all groups were phosphatidylcholine (PC, 48.9 +/- 0.6% of total phospholipid), phosphatidylethanolamine (PE, 33.3 +/- 0.4%), sphingomyelin (SPH, 9.7 +/- 0.3%), phosphatidylserine (PS, 3.5 +/- 0.2%), and phosphatidylinositol (PI, 4.0 +/- 0.5%). No changes in microgram phospholipid/mg membrane protein were observed for any luteal phospholipid on D13 and 15 of the estrous cycle or pregnancy. No significant changes in the relative percentages of major fatty acids present in PC (palmitic [16:0], oleic [18:1]), PE (stearic [18:0], 18:1 and arachidonic [20:4]), or PS (18:0, 18:1, docosatetraenoic [22:4]), nor in the ratios of unsaturated (U) to saturated (S) fatty acids in these phospholipids were observed. Significant differences in unsaturated fatty acids of chain length greater than 20 carbons present in minor quantities in PC, PE, and PS were detected between NP and P ewes as well as between days within reproductive stage. The profile of major fatty acids present in PI revealed decreases in 18:0 and 20:4 in D15-NP and increases in 22:4 and docosapentaenoic acid (22:5) in luteal membranes of both D13- and D15-NP ewes relative to the levels of these fatty acids in PI of corresponding groups of pregnant ewes. There was a general trend for 20:4 levels of PC and PI in membranes of D15-NP ewes to be inversely related to those of D15-P ewes. Collectively, these changes were reflected by an increased U:S fatty acid ratio in luteal membrane PI during the estrous cycle. Specific binding of [125I] iodo-human chorionic gonadotropin to luteal plasma membranes from NP and P ewes on D13 and 15 (6/group) revealed similar affinities and concentrations of unoccupied luteinizing hormone (LH) receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Seminal plasma regulates corpora lutea macrophage populations during early pregnancy in mice

In mice, exposure of the uterus to seminal plasma at mating initiates an inflammatory response within the endometrium, which is characterized by production of cytokines that recruit and activate leukocytes. We hypothesized that this seminal plasma-induced inflammatory response would extend to the ovary, increasing leukocyte abundance within corpora lutea and potentially enhancing progesterone synthesis. Female mice mated to males with their seminal vesicles surgically removed exhibited fewer macrophages within corpora lutea on the day after mating, compared with females mated to vasectomized or normal, intact males. The mean number of F4/80-positive macrophages and major histocompatibility complex (MHC) class II-positive activated macrophages was approximately 2-fold fewer in the absence of seminal vesicle fluid. The effects of seminal plasma on macrophage abundance subsided by Day 4 and were not accompanied by a change in serum progesterone levels during luteinization (Days 1, 2, or 4 after mating) or luteolysis (Days 6 or 9). In vitro secretion of progesterone from corpora lutea cultured with or without LH also did not differ between treatment groups. There was no effect of seminal plasma deficiency in males on the number of ovulated ova or corpora lutea in females. These results imply that seminal plasma exposure of the female reproductive tract at mating augments the macrophage population of newly formed corpora lutea, although these additional macrophages seem not to play a role in steroidogenesis and may instead be involved in tissue remodeling within corpora lutea.     

Total ascorbate and dehydroascorbate concentrations in porcine ovarian stroma, follicles and corpora lutea throughout the estrous cycle and pregnancy

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood constituents during the estrous cycle and early pregnancy in dairy cows

The objective of this study was to determine if maternal platelet count, white blood cell count or other blood constituents undergo sustained alterations in concentration following fertilization. Blood samples from 17 Holstein females were collected over an 18-d period starting at estrus. Blood was analyzed for levels of platelets, white blood cells, red blood cells, hemoglobin and hematocrit. Results were analyzed for differences between nonpregnant and pregnant groups. Analysis of variance revealed a day-by-group interaction in the platelet count (P<0.01). White blood cell count showed both a day-by-group interaction and a difference between days (P<0.01). Red blood cell count, hematocrit and hemoglobin levels resulted in no significant difference between the two groups (P>0.05). While statistically significant differences were observed in platelet and white blood cell count, neither of these were sustained over a period longer than 2 d.     

Uterine contractile activity in mares during the estrous cycle and early pregnancy

Transrectal ultrasonography was used to quantitate uterine contractile activity during the estrous cycle and early pregnancy in pony mares (nonbred, n = 9; pregnant, n = 16). Continuous 1-min scans of longitudinal sections of the uterine body were videotaped, and uterine activity scores (1=minimal activity, 5=maximal activity) were assigned to each tape segment. There was a tendency (P<0.06) for a main effect of reproductive status (nonbred versus pregnant), a main effect of day (P<0.0001), and a reproductive status by day interaction (P<0.006). Uterine activity scores were higher (P<0.05) in pregnant mares on Days 1, 11, 12, and 17 (Day 0=day of ovulation) than in nonbred mares. Maximal activity in pregnant mares occurred on Days 11 to 14 during the reported period of maximal embryo mobility. Activity scores decreased (P<0.05) between the day prior to and the day of fixation (mean = Day 15) of the embryonic vesicle. Activity scores were maintained at an intermediate level for several days following fixation before declining to minimal levels by 7 d postfixation. A postovulatory decrease (P<0.04) in activity scores was observed in nonbred mares, but not in pregnant mares, between Days 0 and 1 followed by a progressive increase (P<0.03) between Days 2 and 4. Maximal activity in nonbred mares occurred during the late luteal phase (Days 13 to 14), corresponding temporally to the reported onset of luteolysis.     

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.     

Follicular development during early pregnancy and the estrous cycle of the sow

The objective of this study was to monitor and compare follicle populations and follicular development in pregnant and nonpregnant sows from Day 3 to Day 20 after breeding. Twenty-four sows were paired within parity on the day of artificial insemination and were randomly allocated within pair for insemination with either killed (n=12) or live spermatozoa (n=12). All the sows were artificially inseminated with the pooled ejaculate of the same boar. From Day 3 through Day 20 post estrus, ovarian follicles were scanned daily by ultrasonography. Ultrasound images were recorded on videotape and were retrospectively analyzed. Follicles were mapped to indentify the existence of follicular waves. The follicles were then classified as small (< 3 mm), medium (3-5 mm), or large (>/=5 mm). Pregnancy diagnosis was performed on Day 21 by ultrasonography. Pregnant sows maintained a constant proportion of the follicle population in the small, medium and large follicle categories. However, in the nonpregnant sows, the proportion of follicles in the various size categories remained constant until Day 15. Thereafter, the proportion of small follicles decreased (P < 0.05) from Day 15 to 20, and the proportions of medium and large follicles increased (P < 0.05). The predictability of pregnancy status on Day 20 based on follicle populations in any of the 3 follicle categories was low. Moreover, there was no evidence of follicular waves during the estrous cycle or early pregnancy. In conclusion, the proportion of small follicles decreased while medium and large follicle increased from Day 15 through Day 20 of the estrous cycle, but not during a similar stage of pregnancy. This latter finding concurs with follicle recruitment from the pool of small follicles for ovulation following PGF2alpha secretion to induce luteolysis, which reduces progesterone concentrations and thereby allows for the stimulation of the pool of small follicles by gonadotropins.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep

Plasma concentrations of oxytocin and progesterone have been measured by radioimmunoassay in jugular venous blood obtained daily from 5 sheep during 2 estrous cycles and in early pregnancy.Concentrations of oxytocin were relatively high (15–30 pg/ml) during the luteal phase of the cycle, but fell at estrus (to 1–17 pg/ml). A fall in oxytocin was also observed on day 15 of pregnancy, when, as expected, progesterone levels remained high. It is suggested that raised basal levels of oxytocin are unlikely to cause the increasing uterine release of prostaglandin F_2α which occurs at the end of the estrous cycle.     

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)     

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.     

Physical characteristics of the uterus during the bovine estrous cycle and early pregnancy

Uterine tone, uterine contractility and endometrial echotexture were monitored daily in heifers during the estrous cycle (n = 6; Days 0 to 21; ovulation = Day 0) and during early pregnancy (n = 7; Days 0 to 26). Uterine tone was assessed by transrectal palpation and scored from 1 (flaccid) to 5 (turgid) by an operator who had no knowledge of reproductive status, day, or group. The main effect of day was significant, but the group effect and the group-by-day interaction were not. Uterine tone scores were high during the periovulatory period (Days--1, 0, 1), decreased (P < 0.05) to low levels on Days 3 and 4, and then increased (P < 0.05) from Days 4 to 10. The increase in tone during early diestrus was confirmed (P < 0.05) in a second experiment. Uterine contractility was assessed by transrectal ultrasonography during a five-minute scan of the caudal portions of the uterine horns and scored from 1 (minimal contractility) to 4 (maximal contractility). The main effects of day and the group-by-day interaction were significant. Contractility scores in both groups were highest just before or on the day of ovulation (Days--1,0) and then decreased (P < 0.05) until Day 11. After Day 16, the scores increased (P < 0.05) in the nonbred heifers and remained low in the pregnant heifers. Endometrial echotexture scores were different among days (P < 0.0001), between the 2 groups (P < 0.02), and for the group-by-day interaction (P < 0.0001). Echotexture scores in both groups peaked just before ovulation (Day--1) and then decreased (P < 0.05) until Day 4. After Day 16, the scores increased in the nonbred group but remained low in pregnant heifers. In summary, uterine contractility and endometrial scores had similar profiles, being high during the periovulatory period and low thereafter; the levels rose in association with the end of the interovulatory interval in nonbred heifers, but remained at low levels in pregnant heifers. Uterine tone scores were also high during the periovulatory period and decreased to low levels several days postovulation, but then, in contrast with the other end points, began to increase in both the nonbred and pregnant heifers.