Nitric oxide induces apoptosis in bovine luteal cells

We previously showed in in vivo and in vitro studies that nitric oxide (NO) is engaged in luteolysis in cattle. Nitric oxide produced locally in the bovine corpus luteum (CL) inhibits progesterone (P4) synthesis and is suggested to be a component of the luteolytic cascade induced by uterine prostaglandin (PG) F2alpha. In the present study, the molecular mechanisms of NO action during structural luteolysis were studied in cultured bovine luteal cells (Days 15-17 of the estrous cycle). The effects of the NO donor (NONOate; 10(-4)M) on DNA fragmentation, cell viability, P4 production and caspase-3 activity were compared with those of PGF2alpha (10(-6)M). Moreover, mobilization of intracellular calcium [Ca2+]i and gene expressions of Fas-L, Fas, bcl-2, bax, and caspase-3 in the cells were determined by semi-quantitative RT-PCR after NONOate treatment. Caspase-3 activity was examined calorimetrically. Contrary to PGF2alpha NONOate decreased cell viability. DNA fragmentation after NONOate treatment increased by more than with PGF22alpha. NONOate increased mobilization of [Ca2+]i in the cells. Although the NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and bax mRNA and caspase-3 expression. The ratio of bcl-2 to bax mRNA level decreased in the cells treated with NONOate. Moreover, NONOate stimulated caspase-3 activity more effectively than PGF2alpha. The overall results suggest that NO is a luteolytic factor that plays a crucial role in regulation of the estrous cycle in structural luteolysis by inducing apoptosis of luteal cells in cattle.     

Effects of nitric oxide and tumor necrosis factor-alpha on production of prostaglandin F2alpha and E2 in bovine endometrial cells

The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)alpha influence on the bovine endometrium. TNFalpha influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFalpha might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFalpha- and NO-regulated prostaglandin (PG) F(2alpha) and PGE(2) synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 microM) for 24 h. S-NAP strongly stimulated PGE(2) production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF(2alpha) production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFalpha and NO action, stromal cells were incubated for 24 h with TNFalpha or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 microM) or an inhibitor of phosphodiesterase (IBMX; 10 microM). When the cells were exposed to TNFalpha in combination with NOS inhibitor (L-NAME), TNFalpha-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFalpha on PGs production (P<0.05). The overall results suggest that TNFalpha augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE(2) production in epithelial cells.     

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO₂/NO₃) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17β-estradiol (E₂; 10⁻⁹ M) and/or progesterone (P₄; 10⁻⁷ M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻⁵ M). Prostaglandin F_2α and PGE₂ secretion was measured in medium by ELISA. The pretreatment of cells with P₄ (progesterone), E₂ (17 β-estradiol), or E₂/P₄ augmented TNF-α-induced PGF_2α and PGE₂ secretion (P < 0.01). The pretreatment of cells with E₂ or E₂/P₄ increased NONOate-induced PGF_2α and PGE₂ secretion (P < 0.01). TNF-α induced NO₂/NO₃ production by BOECs. The pretreatment of cells with E₂ augmented only TNF-α-induced NO₂/NO₃ production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻⁵ M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.     

The effects of estrogen and progesterone on prostaglandins and integrin beta 3 (beta3) subunit expression in primary cultures of bovine endometrial cells

In cattle, endometrial expression of integrin alphavbeta3 is reduced on day 16 of the estrous cycle, coinciding with the critical period during which the decision is made to initiate luteolysis or continue with pregnancy. The objective of these experiments was to examine the relationship between estrogen and progesterone treatments, endometrial integrin alphavbeta3 expression, and prostaglandin F2alpha (PGF2alpha) and E2 (PGE2) production. Epithelial and stromal cells from intercaruncular (ICAR) and caruncular (CAR) bovine endometrium were treated with 17beta-estradiol (0.1 and 1.0 nM) and/or progesterone (1.0 and 10 nM) in a manner designed to mimic the steroid fluctuations of the estrous cycle. All cell types expressed estrogen receptor and progesterone receptor mRNA and protein. Intercaruncular stromal cells were the most responsive to steroidal regulation. Estrogen suppressed expression of integrin subunit beta3 mRNA in ICAR stromal cells (P< or =0.05). Progesterone and estrogen + progesterone treated cells did not differ in beta3 expression from controls (P> or =0.05). Steroid treatment did not affect PGF2alpha production in any cell type (P> or =0.05), however, estrogen decreased PGE2 production in all cells except CAR stroma (P< or =0.05). The results indicate that in bovine endometrium expression of integrin alphavbeta3 and production of PGE2 is influenced by estrogen.     

Role of tumor necrosis factor-alpha and nitric oxide in luteolysis in cattle

Although prostaglandin (PG) F2alpha is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-alpha (TNFalpha) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFalpha in combination with interferon-gamma reduced progesterone (P4) secretion, increased PGF2alpha and leukotriene C4 (LTC4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFalpha caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF2alpha, LTC4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF2alpha-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF2alpha and TNFalpha actions during luteolysis. Both PGF2alpha and TNFalpha increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF2alpha and LTC4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFalpha and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFalpha and NO play crucial roles in functional and structural luteolysis in cattle.     

Contractile effect of PGF2alpha and PGE2 on isolated branches of uterine and ovarian artery in different days of estrous cycle and early pregnancy in pigs

The contractile effects of PGF2alpha (3 x 10(-6) to 10(-4) M) and PGE2 (10(-7) to 10(-5) M) were examined on isolated branches of ovarian artery (OA) and extramyometrial branches of uterine artery (UA) collected from pigs in the luteal (day 10-12) and follicular phase (day 17-20) of the estrous cycle, and during early pregnancy (day 10-12). Strong contraction was demonstrated in both arteries during all investigated periods in response to PGF2alpha, which was significantly higher (P < 0.01) than to PGE2, being negligible in the follicular phase. In UA, the effective dose of PGF2alpha (ED50) amounted 7.9 x 10(-6) M and 6.3 x 10(-6) M in the luteal and follicular phase, and 5.0 x 10(-6) M in early pregnancy. ED50 for PGE2 reached 5.0 x 10(-7) M in the luteal phase, and 4.1 x 10(-7) M in early pregnancy. For both prostaglandins, the contraction was much stronger (P < 0.01) in OA than in UA branches. In OA, the ED50 for PGF2alpha was 1.2 x 10(-5) M in the luteal phase and was significantly higher (P < 0.05) than in the follicular phase (3.1 x 10(-6) M) and early pregnancy (2.7 x 10(-6) M). ED50 for PGE2 amounted 7.3 x 10(-7) M in the luteal phase and 1.7 x 10(-7) M in early pregnancy. Studies showed the influence of the estrous cycle and early pregnancy on OA branches sensitivity to the contractile effect of PGF2alpha and the lack of this effect on UA branches, and the influence of the estrous cycle on UA and OA branch contraction in response to PGE2.     

Tumor necrosis factor-α inhibits the stimulatory effect of luteinizing hormone and prostaglandin E(2) on progesterone secretion by the bovine corpus luteum

Tumor necrosis factor-α (TNF-α) is involved in the tissue remodeling that occurs in the corpus luteum (CL) during its development and regression. This cytokine is also implicated in the regulation of reproduction by its actions on ovarian steroidogenic cells. The aim of this study was to examine the influence of TNF-α on (1) progesterone (P(4)) output by the bovine CL and on (2) the responsiveness of the CL to LH or prostaglandin E(2) (PGE(2)) in vitro. In experiment 1, CL (days 8 to 10 of the estrous cycle) were perfused by using an in vitro microdialysis system with TNF-α (0.1, 0.5, or 1 μg/mL) alone or with TNF-α (1 μg/mL) followed by LH (1000 ng/mL) or PGE(2) (2 × 10(-5) M). Basal P(4) release (P < 0.05) was increased by TNF-α (0.5 or 1 μg/mL). Moreover, TNF-α (1 μg/mL) inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). In experiment 2, 4 h after intrauterine infusion of TNF-α (0.01 μg/mL or 1 μg/mL), CL (days 8 to 10 of the estrous cycle) were collected by colpotomy, cultured, and stimulated with LH (10 ng/mL) or PGE(2) (10(-6) M). Intrauterine infusion of TNF-α at a concentration of 1 μg/mL increased basal P(4) output by CL (P < 0.05). Moreover, the intrauterine infusion of TNF-α at a concentration of 0.01 μg/mL inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). These results indicate that TNF-α (1) does not have an effect on the autonomous, pulsatile release of P(4); (2) increases P(4) secretion by bovine CL with increasing doses, and (3) reduces in a dose-dependent manner the responsiveness of CL to luteotropic factors both directly (after infusion to CL) and indirectly (after intrauterine infusion).     

Role of Tumour Necrosis Factor α in Stimulation of Prostaglandins F2α and E2 Release by Cultured Porcine Endometrial Cells

The present studies were undertaken to examine the effect of tumour necrosis factor (TNF) alpha on prostaglandins (PGs) F(2alpha) and E(2) release by cultured porcine endometrial cells harvested on days 13-16 after oestrus in comparison to stimulation with oxytocin (OT) and luteinizing hormone (LH). A time-dependent effect of TNFalpha (10 ng/ml) on PGF(2alpha) release was observed in stromal and luminal epithelial cells. Moreover, TNFalpha increased PGF(2alpha) secretion from both endometrial cell types with effective concentrations of 1 (p < 0.05), 10 and 50 ng/ml (p < 0.01). The effect of TNFalpha (10 ng/ml) on endometrial PGF(2alpha) and PGE(2) release was compared with OT (100 nmol/l) and LH (100 ng/ml). All factors affected PGF(2alpha) secretion from stromal cells, however, the stimulation tended to be more potent after OT and LH (p < 0.01) than after TNFalpha (p < 0.05) treatment. In epithelial cells, only TNFalpha was able to stimulate PGF(2alpha) release (p < 0.001). PGE(2) secretion from stromal cells increased after incubation with TNFalpha and OT (p < 0.05). Only LH stimulated PGE(2) release from epithelium (p < 0.001), and its action was very effective when compared with TNFalpha or OT (p < 0.01). Summarizing, TNFalpha induces both PGs secretion from cultured porcine endometrium, but preferentially stimulates PGF(2alpha) release from luminal epithelial cells. Therefore, similarly to OT and LH, TNFalpha may be considered as a potential modulator of endometrial PGF(2alpha) production during luteolysis in the pig.     

Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production

To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.     

Role of interleukin-1β in the regulation of porcine corpora lutea during the late luteal phase of the cycle and during pregnancy

Interleukin-1β (IL-1β) may regulate ovarian physiology. In this study, the influence of IL-1β on secretory activity within the corpora lutea (CL) of cyclic and gravid pigs was determined in vitro during different stages of the CL lifespan, e.g. on Days 10-11, 12-13 and 15-16 of the oestrous cycle and pregnancy. IL-1β (10 ng/ml) increased prostaglandin E2 (PGE2) secretion from CL of the cyclic and gravid pigs during studied days of the oestrous cycle and pregnancy. Increase (P < 0.05) of prostaglandin F2α (PGF2α) in IL-1β-treated CL was demonstrated only on Days 10-11 of the oestrous cycle. More potent stimulatory effect of IL-1β on PGE2 than PGF2α secretion resulted in the enhancement of the PGE2:PGF2α ratio in cyclic and early pregnant CL. IL-1β increased (P < 0.05) progesterone (P4) secretion only in gravid CL and had no effect on oestradiol-17β (E2) release. Expression of cyclooxygenase-2 (COX-2) mRNA was stimulated (P < 0.05) in IL-1β-treated cyclic and gravid CL. Expression of prostaglandin synthase mRNAs in response to IL-1β did not increase. In conclusion, IL-1β modulates PGE2, PGF2α and P4 secretion from porcine CL, depending on luteal stage and the surrounding hormonal milieu. The cytokine may act locally in porcine CL for luteotrophic support throughout the PGE2-mediated synthesis and secretion.     

A passage and storage system for isolated bovine endometrial epithelial and stromal cells

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.     

Prostaglandins F2α and E2 Secretion by Porcine Epithelial and Stromal Endometrial Cells on Different Days of the Oestrous Cycle

The endometrial tissue of the uterus plays a key role in reproduction and is a source of hormones and factors responsible for the proper physiological function of reproductive tract during the oestrous cycle and pregnancy. In this study, we investigated the pattern of PGF(2alpha) and PGE(2) secretion from cultured porcine endometrial cells at different days of the oestrous cycle. Epithelial and stromal cells were isolated by differential enzymatic digestion on days 6-8, 10-12 and 14-16. After attachment cells were incubated for 3 and 24 h to estimate PGF(2alpha) and PGE(2) output. The purity of culture was 85-90% for epithelial and 95-98% for stromal cells as determined by immunofluorescent staining. Release of PGF(2alpha) and PGE(2) was affected by cell type, days of the oestrous cycle and the time of incubation. After 3 h of incubation epithelial cells secreted more PGF(2alpha) than PGE(2) during all studied periods of the oestrous cycle (p < 0.01 and p < 0.001, respectively), whereas stromal cells released more PGE(2) (p < 0.01) on days 10-12 and 14-16. Longer incubation of stromal cells revealed that PGF(2alpha) output tended to overcome PGE(2) on days 10-16. The lowest secretion of prostaglandins was observed on days 6-8 in both cell types. The highest secretion of PGF(2alpha) from epithelium was measured on days 10-12 after 24 h of incubation when compared with other days studied (p < 0.001). In stromal cells, PGE(2) output increased on consecutive days studied (p < 0.001) after 3 h of incubation. The differential properties of endometrial cell types seem to play an important role in the profile of PGF(2alpha) and PGE(2) release before and during luteolysis. Described endometrial cells culture might serve as the model for further studies on the hormonal regulation of prostaglandin production in the pig.     

The influence of nitric oxide on the contractile activity of the isolated porcine ovarian and uterine arteries

The aim of the present study was to investigate the influence of nitric oxide (NO) on the contractile activity of the isolated porcine ovarian and uterine arteries. Segments of the vessels, obtained from the pigs on days 1-5, 8-13 and 17-20 of the oestrous cycle, were mounted in the organ bath with Krebs-Ringer solution and contractile activity changes of the vessels were measured using isometric transducers. In Experiment I the arteries pretreated with norepinephrine (NE; 10(-7) M) were treated with sodium nitroprusside (SNP, 10(-8)-10(-4) M), a NO donor. In Experiment II administration of NE (10(-7) M) was preceded by treatment with Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-8)-10(-6) M), an inhibitor of NO synthase. Donor of NO at doses of 10(-8)-10(-7) M did not affect (P>0.05) the contractility, while at doses of 10(-5)-10(-4) M caused a dose-dependent relaxation (P<0.05) of both ovarian and uterine arteries in all periods examined. Moreover, SNP at doses of 10(-6)-10(-4) M it caused significantly higher (P<0.05) relaxation of the ovarian arteries collected on days 8-13 as compared to the vessels from days 1-5 of the cycle. Pretreatment of the vessels with L-NAME caused a dose-dependent, significant (P<0.05) increase in the vasocontractile action of NE in both the ovarian and uterine arteries as compared to contractile activity of NE administered alone. Moreover, L-NAME pretreatment at a dose of 10(-6) M caused significantly higher (P<0.05) intensification of NE action in ovarian and uterine arteries collected on days 8-13 as compared to the vessels from days 1-5 (P<0.05) and 17-20 (P<0.05) of the oestrous cycle. Obtained results indicate that NO plays an important role in the regulation of the contractile activity of the isolated porcine ovarian and uterine arteries. Our data suggest that this action may be, at least in a part, dependent on the hormonal status of the organism.     

Regulation of mitogen- and TCGF-induced lymphocyte blastogenesis by prostaglandins and supernatant from equine embryos and endometrium

Immunosuppressive substances which interfere with lymphocyte blastogenesis are released in vitro by embryos and endometrium from mares in early pregnancy. Immunosuppression was not evident when tissues were cultured in the presence of indomethacin (a prostaglandin-synthesis inhibitor). Various prostaglandins (PGs) were added to equine lymphocytes and lymphocyte proliferation was measured after the addition of concanavalin A (Con A) or phytohaemagglutinin A (PHA). PGE2 and PGF2 alpha inhibited Con A-induced blastogenesis down to final concentrations of 1.8 x 10(-9) M and 1.3 x 10(-6) M, respectively. Other PGs tested (6-keto-PGF1 alpha and 13,14-dihydro-15-keto-PGF2 alpha) did not affect Con A-induced blastogenesis. PHA-induced blastogenesis was inhibited by concentrations down to 1.8 x 10(-9) M PGE2, 3.3 x 10(-7) M PGF2 alpha and 2.8 x 10(-4) M 6-keto-PGF1 alpha. At all concentrations, 13,14-dihydro-15-keto-PGF2 alpha only slightly reduced PHA-induced blastogenesis. Therefore, PGE2 was the only prostaglandin tested which, at physiological concentrations, significantly inhibited incorporation of [3H] thymidine. The mechanism of PGE2-mediated suppression was studied by adding PGE2 and T cell growth factors (TCGF) to TCGF-responsive lymphocytes. PGE2 reduced the TCGF-mediated blastogenic response in a dose-dependent manner. Furthermore, culture supernatant from embryos and endometrium from 14-day pregnant mares inhibited lymphocyte blastogenesis induced by TCGF. These results show that PGE2 interferes with lymphocyte blastogenesis by TCGF-dependent mechanisms. It is suggested that the PGE2 present in the uterus of the early pregnant mare may be one of the factors involved in immunosuppression at the time of maternal recognition of pregnancy.     

Expression of mRNAs for interleukin-4, interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle

There is increasing evidence that inflammatory cytokines regulate corpus luteum (CL) function in many species. The purpose of the present study was to determine whether interleukin (IL)-4 and IL-6 are expressed in the porcine CL, and whether these cytokines influence porcine luteal steroidogenesis. The gene expressions of IL-4, IL-6 and their specific receptors were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of these cytokines on progesterone (P(4)), estradiol-17beta (E(2)) and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. IL-4 and IL-6 mRNAs were detected in the CL at all luteal stages. Furthermore, mRNAs of the receptors for IL-4 and IL-6 were clearly expressed in the CL throughout the estrous cycle. Real-time PCR analysis revealed that IL-6 receptor (IL-6R) mRNA expression was higher in the regressed CL (days 19-21 after ovulation) than in the CL at other stages (P<0.01). Exposure of cultured luteal cells obtained from mid-stage CL (days 8-11) to IL-6 (1-100 ng/ml), it inhibited P(4) and E(2) secretion by the cells (P<0.05). Although IL-4 (1-100 ng/ml) did not significantly alter P(4) secretion, it inhibited E(2) secretion by the cells (P<0.05). Neither IL-4 nor IL-6 had any effect on PGF2alpha secretion by the cells. These results suggest that IL-4 and IL-6 are locally produced in the porcine CL, and that they inhibit steroid production from luteal cells via their specific receptors. Collectively, both IL-4 and IL-6 may play roles in regulating porcine CL function throughout the estrous cycle.     

Prostaglandin E2-9-ketoreductase activity of preovulatory ovine follicles

Enhanced accumulation of follicular PGF2 alpha with respect to PGE2 during the later phase of the preovulatory period is an apparent prerequisite for ovulation in sheep. Prostaglandin (PG) E2-9-ketoreductase is the enzyme that converts PGE2 into PGF2 alpha. Expression of activity of this enzyme by tissue homogenates of preovulatory ovine follicles was assessed. Homogenates were incubated in the presence of tritiated PGE2. Prostaglandin F2 alpha (i.e., product) was separated from PGE2 by Sephadex chromatography and quantitated by liquid scintillation counting. Progesterone in follicular fluid was measured by RIA. Follicular activity of PGE2-9-ketoreductase and content of progesterone increased approximately sixfold as the time of ovulation approached. Formation of PGF2 alpha from PGE2 was not influenced by inhibition of follicular synthesis of prostaglandins by indomethacin, nor did such treatment affect follicular production of progesterone. Inhibition of follicular synthesis of progesterone by isoxazol suppressed enzymatic conversion of PGE2 into PGF2 alpha; this effect was reversed by progesterone. It appears that progesterone plays an intrafollicular role in induction of activity of PGE2-9-ketoreductase in sheep.     

Induction of ovulation with GnRH and PGF(2 alpha) at two different stages during the early postpartum period in dairy cows: ovarian response and changes in hormone concentrations

The aims of this study were 1) to determine whether dairy cows can be induced to ovulate by the treatment with gonadotropin releasing hormone (GnRH) followed by prostaglandin F(2 alpha) (PGF(2 alpha)) during the early postpartum period and 2) to describe their ovarian and hormonal responses according to ovarian status. Cows were divided in two groups and received 10 microg of buserelin followed by 500 microg of cloprostenol 7 days apart starting from 21 (GnRH21, n=7) or around 37 days postpartum (GnRH37, n=7). The groups were further classified according to presence (-CL) or absence (-NCL) of functional corpora lutea (CL) on the day of GnRH treatment (d 0): GnRH21-NCL (n=4), GnRH21-CL (n=3) and GnRH37-CL (n=7). Ovarian morphology was monitored and the concentrations of P(4), E(2), FSH and insulin-like growth factor 1 (IGF-1) were measured. All cows ovulated after administration of GnRH. The P(4) levels of the GnRH21-NCL group from d 0 to d 5 were lower than those of the GnRH21-CL (P<0.05) and GnRH37-CL groups (P<0.01). In contrast, the E(2) levels of the GnRH21-NCL group within d 2 to d 6 were higher (P<0.05) than those of the other groups. Compared with the GnRH37-CL group, the GnRH21-NCL group had more small follicles on d 2 (P<0.05), d 3 (P<0.01) and d 4 (P<0.01) and more large follicles on d 5 (P<0.05). The induced CL and new ovulatory follicles were larger in the GnRH21-NCL group compared with the GnRH21-CL (P<0.001 and P<0.01) and GnRH37-CL groups (P<0.001 and P<0.05). IGF-1 did not differ among the groups. The GnRH21-NCL group had higher FSH levels than the GnRH21-CL (P<0.01) and GnRH37-CL groups (P<0.001) on d 0. Low P(4) and high FSH levels may suggest higher gonadotropin support on the enhanced ovarian morphology of the GnRH21-NCL group. PGF(2 alpha) treatment induced CL regression and subsequent ovulation in 3/4 (75%), 3/3 (100%) and 7/7 (100%) cows in the GnRH21-NCL, GnRH21-CL and GnRH37-CL groups, respectively. In conclusion, a 7-day GnRH-PGF(2 alpha) synchronization protocol can effectively induce dairy cows to ovulate as early as 21 days postpartum, regardless of ovarian status.     

Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.