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.     

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.     

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.     

Immunohistochemical localization of estrogen receptors in the ovine corpus luteum throughout the estrous cycle

Using immunohistochemistry and Western blot analysis we attempted to identify the estrogen receptors in ovine luteal cells at different stages of the estrous cycle. Monoclonal antibody against estrogen receptors was used for immunolocalization of estrogen receptor-alpha in corpora lutea sections. Generally, the most intense cytoplasm staining was present in large luteal cells. On the 6th day of the estrous cycle, weak immunostaining of estrogen receptors was observed in large luteal cells as well as in the connective tissue. Luteal cells from regressing corpora lutea expressed the weakest immunostaining. The most intense immunoreactivity for estrogen receptors was found in sections of corpora lutea collected on the 9th day of the cycle. Both, cytoplasmic and nuclear localization was observed depending on cell types in the ovine corpus luteum. Our studies demonstrated the presence of the estrogen receptor-alpha in the luteal cells and suggested an autocrine/paracrine role of estrogen in the regulation of estrous cycle in sheep.     

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.     

Growth and cellular proliferation of pig corpora lutea throughout the oestrous cycle

Corpora lutea were obtained from gilts on days 2, 4, 8, 12, 15 or 18 after oestrus. Luteal fresh masses and DNA contents increased linearly (P < 0.01) from day 2 to day 12 and day 2 to day 15, respectively. Changes in the ratio of protein:DNA were greatest between days 2 and 4 and days 15 and 18, whereas changes in DNA content were relatively small during the same intervals. Thus, a major component of changes in the size of the corpus luteum during the early and late periods of the luteal phase was cellular hypertrophy. Proliferation of luteal cells in vivo (nuclear incorporation of 5-bromo-2-deoxyuridine, a thymidine analogue) was greatest on day 2 and decreased exponentially (P < 0.01) throughout the oestrous cycle. Results from co-localization of 5-bromo-2-deoxyuridine and factor VIII (von Willebrand factor), a marker of endothelial cells, or 5-bromo-2-deoxyuridine and 3 beta-hydroxysteroid dehydrogenase, a marker of steroidogenic cells, indicated that some of the luteal steroidogenic cells proliferated early in luteal development. However, during early and mid-cycle, most of the luteal cell proliferation occurred in the endothelial cells. Thus, during growth of the pig corpus luteum, which is extremely rapid, most of the proliferating luteal cells are vascular endothelial cells. This observation is consistent with the high vascularity and blood flow of the mature corpus luteum and implies a critical role for angiogenesis in luteal development in the pig, as has been proposed for several other mammalian species.     

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.     

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).     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E2 (PGE2), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F2alpha (PGF2alpha) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.     

Expression and localization of transient receptor potential channels in the bovine uterus epithelium throughout the estrous cycle

Transient receptor potential (TRP) channels are expressed in the endometrium but it is unknown if they are modulated through the estrous cycle (EC). This study was undertaken to identify the modulation of the TRPC gene and protein isoforms in bovine uterine epithelium, as a model for human, throughout the EC. Changes in the expression of TRPC genes in bovine uterine epithelium throughout the EC were measured using Real-Time PCR, while immunohistochemistry and immunocytochemistry were used to determine the localization of these channels. Out of the 7 members of the TRPC family, TRPC1, 2, 3, 4 and 6 genes were expressed in bovine uterine epithelial tissue and TRPC 5 and 7 were not. Gene expression levels of all TRPC isoforms underwent cyclical changes throughout the EC. Moreover, cyclical changes were detected in the protein levels of TRPC1 and TRPC6 throughout the EC. These findings show that TRPC channels are modulated through the EC and therefore may have a role in reproductive events.

     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E(2) (PGE(2)), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F(2alpha) (PGF(2alpha)) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P >/= 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P /= 0.05) while PGE(2) increased (P /= 0.05) detectable quantities of PGF(2alpha) or PGE while day-90 ovine CL of pregnancy secreted PGE (P /= 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF(2alpha) by day-11 CL of the estrous cycle or day-90 CL of pregnancy (P >/= 0.05). It is concluded that PGE(2), not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF(2alpha).     

Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-oligoadenylate synthetase expression in the ovine uterus

The enzymes which comprise the 2',5'-oligoadenylate synthetase (OAS) family are interferon (IFN) stimulated genes which regulate ribonuclease L antiviral responses and may play additional roles in control of cellular growth and differentiation. This study characterized OAS expression in the endometrium of cyclic and pregnant ewes as well as determined effects of IFNtau and progesterone on OAS expression in cyclic or ovariectomized ewes and in endometrial epithelial and stromal cell lines. In cyclic ewes, low levels of OAS protein were detected in the endometrial stroma (S) and glandular epithelium (GE). In early pregnant ewes, OAS expression increased in the S and GE on Day 15. OAS expression in the lumenal epithelium (LE) was not detected in uteri from either cyclic or pregnant ewes. Intrauterine administration of IFNtau stimulated OAS expression in the S and GE, and this effect of IFNtau was dependent on progesterone. Ovine endometrial LE, GE, and S cell lines responded to IFNtau with induction of OAS proteins. In all three cell lines, the 40/46-kDa OAS forms were induced by IFNtau, whereas the 100-kDa OAS form appeared to be constitutively expressed and not affected by IFNtau. The 69/71-kDa OAS forms were induced by IFNtau in the S and GE cell lines, but not in the LE. Collectively, these results indicate that OAS expression in the endometrial S and GE of the early pregnant ovine uterus is directly regulated by IFNtau from conceptus and requires the presence of progesterone.