Luteal mRNA Research Articles

221 Putative Gene Regulators in the Equine Endometrium, Corpus Luteum and Embryo During Early Pregnancy in the Horse

Abstract It is estimated that 17.3% of all pregnancies in the horse are lost from the time of detection to day 40, which has significant economic impact. But still, equine reproductive research has limited funding opportunities. Understanding the dynamics between the embryo, endometrium, and corpus luteum (CL) is essential to ameliorate those losses. Pig embryonic interferon (IFN) gamma is essential for the establishment of pregnancy. In ruminants, IFN tau is the maternal recognition signal. The equine embryo seems to express IFN alpha, gamma, delta, and epsilon but higher expression was only identified on day(D) 16 embryos, after the maternal recognition of pregnancy (MRP), so the IFN role during pregnancy is unknown. Oxytocin and prostaglandins have been demonstrated to have a role during luteolysis and luteostasis in the horse, and therefore investigation the embryonic, endometrium, and CL putative gene regulators during the equine MRP is warranted. Oxytocinase (LNPEP), an aminopeptidase that metabolizes oxytocin (OXT) has been recently investigated with other putative gene regulators in the horse during the expected time of MRP. OXT and LNPEPmRNA were identified in the equine embryo, endometrium, and CL, but OXT/neurophysin-1 protein was not detected in the embryo, and present in the CL. Most of the endometrial and luteal mRNA differences were identified around D12 post-ovulation, suggesting being a critical time for MRP. Luteal OXT was not different between pregnant and non-pregnant mares. LNPEP mRNA was upregulated in pregnant and non-pregnant mares D10 and 12 luteal tissues along with prostaglandin E synthase (PTGES). Embryonic trophoblast LNPEP and PTGES have higher relative abundance on D15 conceptus compared earlier embryos, suggesting a potential role during embryonic fixation. The increased expression of LNPEP is suggested to have a role in decreasing oxytocin availability and shifting the ratio PGE:PGF to a PGE that will support luteal function during early pregnancy.

Endometrial and luteal gene expression of putative gene regulators of the equine maternal recognition of pregnancy

Our understanding of the temporal changes in endometrial and luteal gene transcripts related to the actions of oxytocin and prostaglandin during early equine pregnancy is incomplete. Additionally, the role of oxytocinase, also known as Leucyl-cystinyl aminopeptidase (LNPEP), during early pregnancy in mares has not been previously investigated. Luteal and endometrial biopsies were obtained on Day (D)8, D10, D12 and D15 post-ovulation in pregnant (PREG) and diestrus (DIEST) mares for real-time qPCR. Differences in endometrial gene expression occurred over time in: SLC2A4, SLC2A1, PTGES, OXTR and LNPEP. PTGFR and PLA2G2C had lower relative abundance in PREG D15 endometrium compared to D10. OXT and OXTR were increased on D10 and 15 PREG, respectively. Regarding luteal mRNA relative abundance, ESR1, PTGS2, PTGFR, and PTGES had higher relative abundance in D12 of DIEST and PREG. Luteal expression of OXTR and OXT had higher relative abundance in D15 compared to D8, and LNPEP had higher relative abundance in D10 and 12. Endometrial and luteal PTGES had an increased mRNA abundance in both D12 DIEST and PREG mares, which may lead to additional luteoprotective prostaglandin E2 (PGE2) secretion. Furthermore, luteal SLC2A1 had higher relative abundance in pregnancy, and likely supports the high metabolic activity of luteal tissue by increasing glucose uptake. Oxytocinase is present in endometrial and luteal tissue and its role in oxytocin induced prostaglandin secretion is uncertain.

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5mg, respectively) during mid-luteal phase (12 and 10days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Expression profile of the Kisspeptin/Kiss1r system and angiogenic and immunological mediators in the ovary of cyclic and pregnant cats

The Kisspeptin/Kiss1r system has been studied in mammalian ovaries. However, there are still no studies on the modulation of this system and its relationship with angiogenic and immunological mediators in the ovary of domestic cats, especially during pregnancy. We evaluated the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators during folliculogenesis, luteogenesis and luteal regression of cyclic and pregnant cats. The ovary exhibited moderate to intense expression for Kiss1, VEGF, Flk-1, INFγ and MIF in oocytes and the follicular wall, while Kiss1r expression was low in granulosa cells. In these cells, there was also a greater expression of Kiss1, INFγ and MIF, mainly in secondary follicles, while tertiary and preovulatory follicles exhibited greater expression of VEGF and Flk-1 in this layer. In luteogenesis, Kiss1 immunostaining was higher in mature corpora lutea (MCL) of pregnant cats compared to vacuolated CL (VCL) and corpus albicans (CA). Pregnancy also increased the luteal gene expression of Kiss1 as well as Kiss1, Kiss1r, Flk-1, and MIF immunostaining in MCL, while reduced the area of VEGF expression in VCL and luteal mRNA expression of Mif when compared to non-pregnant animals. In addition, positive gene correlation between Kiss1r and Mif was observed in the CL. Kiss1, Kiss1r, Vegf and Mif expression were lower in the CA of cats in anestrus. These findings reveal that the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators, in the ovary of domestic cats, depend on the follicular and luteal stage, and the luteal expression of these mediators is influenced by pregnancy.

Effects of LPA2R, LPA3R, or EP4R agonists on luteal or endometrial function in vivo or in vitro and sirtuin or EP1R, EP2R, EP3R or EP4R agonists on endometrial secretion of PGE and PGF2αin vitro

In previous work, an EP2 prostanoid receptor (EP2R) agonist in vivo increased mRNA expression of luteal LH receptors (LHR), unoccupied and occupied luteal; LHR, and circulating progesterone, while an EP3R or FPR agonist decreased; mRNA expression of luteal LHR, unoccupied and occupied luteal LHR, and; circulating progesterone. An EP4R and lysophosphatidic acid (LPA) LPA2R and LPA3R agonists were reported to inhibit luteal function and sirtuins have been proposed to increase prostaglandin synthesis. The objectives were to determine; whether an EP4R, LPA2R, or LPA3R agonist affect ovine luteal function in vivo or; in vitro. In addition, whether sirtuin (SIRT)-1, 2, or 3; LPA2R or LPA3R; or EP1R, EP2R, EP3R, or EP4R agonists affect caruncular endometrial PGF2α or PGE (PGE1+PGE2) secretion in vitro. Day-10 nonpregnant ewes received a single injection of Vehicle (N = 5); an LPA2R (N = 5); LPA3R (N = 6); or EP4R (N = 5) agonist given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary to determine effects on circulating progesterone, mRNA expression of luteal LHR, and luteal unoccupied and occupied LHR. In addition, agonists for LPA2R, LPA3R, EP1R, EP2R, EP3R, or EP4R or SIRT-1, SIRT-2, or SIRT-3 activators were incubated with caruncular endometrial slices in vitro to determine their effect on caruncular endometrial PGF2α, or PGE secretion. LPA2R, LPA3R, or an EP4R agonist in vivo did not affect (P ≥ 0.05) luteal weight, circulating progesterone, or occupied luteal LHR. However, an LPA2R or EP4R agonist, but; not LPA3R agonist, in vivo increased (P ≤ 0.05) mRNA expression of luteal LHR. An; LPA2R, LPA3R, or EP4R agonist increased (P ≤ 0.05) luteal unoccupied LHR, but; not occupied LHR. An LPA2R, LPA3R, or an EP4R agonist did not affect (P ≥ 0.05); luteal progesterone secretion in vitro. An LPA2R or LPA3R agonist did not affect (P ≥ 0.05) luteal PGF2α, or PGE secretion in vitro. However, an EP4R agonist tended to decrease (P < 0.066) luteal PGF2α secretion and increased (P ≤ 0.05) luteal PGE; secretion in vitro. EP1R, EP2R, EP3R, or an EP4R agonist did not affect (P ≥ 0.05); caruncular endometrial PGF2α secretion in vitro. However, EP1R, EP3R, or an EP4R agonist increased caruncular endometrial PGE secretion in vitro, while two different EP2R agonists did not affect (P ≥ 0.05) caruncular endometrial PGE; secretion. A SIRT-1 activator, but not SIRT-2 or SIRT-3 activators, increased (P ≤ 0.05) caruncular endometrial PGE secretion, while sirtuin 1, 2, or 3 activators did not affect (P ≥ 0.05) caruncular endometrial PGF2α secretion. In conclusion, receptors for EP4, LPA2, and LPA3 do not appear to be involved; in luteolysis, but EP4R and LPA2R might participate in preventing luteolysis by maintaining luteal mRNA expression for LHR and preventing loss of unoccupied luteal LHR. In addition, SIRT-1, EP1R, EP3R, and EP4R might be involved in; regulating caruncular endometrial PGE secretion, but not PGF2α secretion.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase β protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep.

Repeated intrauterine infusions of lipopolysaccharide alter gene expression and lifespan of the bovine corpus luteum

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9mL) or Escherichia coli lipopolysaccharide (LPS; 3µg/kg of body weight diluted in 9mL of PBS) at 6-h intervals from 12h before and until 9d after ovulation during 2 cycles in a random order (ovulation=d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1β, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum.

Intramammary lipopolysaccharide infusion alters gene expression but does not induce lysis of the bovine corpus luteum

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 μg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

Prolactin modulates luteal regression from the coeliac ganglion via the superior ovarian nerve in the late-pregnant rat.

There is considerable evidence of the neuroendocrine control involved in luteal regression in the rat. In addition, circulating prolactin (PRL), which increases during the night before parturition, may gain access to the coeliac ganglion (CG), indirectly impacting the physiology of the ovary because of the known connection between the CG and the ovary via the superior ovarian nerve (SON). In this work we investigated in the CG-SON-ovary system and whether PRL added to the CG has an impact, indirectly via the SON, on luteal regression on Day 21 of pregnancy. The system was incubated without (control) or with PRL added to the CG. We measured the ovarian release of progesterone (P), oestradiol and prostaglandin F2 alpha (PGF2α) by radioimmunoassay, and nitrites (NO) by the Griess method. Luteal mRNA expression of 3β-hydroxysteroid dehydrogenase (3β-HSD), 20α-HSD, aromatase, inducible nitric oxide synthase (iNOS) and apoptosis regulatory factors was analysed by reverse transcription-polymerase chain reaction. P release, the expression of Bcl-2 and the Bcl-2:Bax ratio was lower than control preparations, while the expression of 20α-HSD and the release of NO and PGF2α were higher in the experimental group. In conclusion, PRL acts at the CG and, by a neural pathway, modulates luteal function at the end of pregnancy.

Effect of prolactin acting on the coeliac ganglion via the superior ovarian nerve on ovarian function in the postpartum lactating and non-lactating rat

Whether prolactin (PRL) has a luteotrophic or luteolytic effect in the rat ovary depends on the nature of the corpora lutea present in the ovaries and the hormonal environment to which they are exposed. The aim was to investigate the effect of PRL acting on the coeliac ganglion (CG) on the function of the corpora lutea on day 4 postpartum under either lactating or non-lactating conditions, using the CG–superior ovarian nerve–ovary system. The ovarian release of progesterone (P), estradiol, PGF2α, and nitrites was assessed in the ovarian compartment at different incubation times. Luteal mRNA expression of 3β-HSD, 20α-HSD, aromatase, PGF2α receptor, iNOS, Bcl-2, Bax, Fas and FasL was analysed in the corpus luteum of pregnancy at the end of the experiments. Comparative analysis of control groups showed that the ovarian release of P, nitrites, and PGF2α, the expression of PGF2α receptor, and the Bcl-2/Bax ratio were lower in non-lactating rats, with increased release of estradiol, and higher expression of aromatase, Fas and FasL, demonstrating the higher luteal functionality in ovaries of lactating animals. PRL added to the CG compartment increased the ovarian release of P, estradiol, nitrites and PGF2α, and decreased the Bcl-2/Bax ratio in non-lactating rats; yet, with the exception of a reduction in the release of nitrites, such parameters were not modified in lactating animals. Together, these data suggest that the CG is able to respond to the effect of PRL and, via a neural pathway, fine-tune the physiology of the ovary under different hormonal conditions.

In vivo intra-luteal implants of prostaglandin (PG) E 1 or E 2 (PGE 1, PGE 2) prevent luteolysis in cows. II: mRNA for PGF 2α, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue

Previously, it was reported that chronic intra-uterine infusion of PGE 1 or PGE 2 every 4 h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE 2 given intra-uterine every 8 h did not inhibit luteolysis in cows, but infusion of estradiol + PGE 2 inhibited luteolysis. In contrast, intra-luteal implants containing PGE 1 or PGE 2 in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE 1 or PGE 2 alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE 1, or PGE 2 and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE 1 and PGE 2 up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE 1 and PGE 2 down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE 1 or PGE 2 up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE 1 and PGE 2 support their agonistic effects. We conclude that both PGE 1 and PGE 2 may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.

In vivo intra-luteal implants of prostaglandin (PG) E 1 or E 2 (PGE 1, PGE 2) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH

Previously, it was reported that chronic intra-uterine infusion of PGE 1 or PGE 2 every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE 2 given intra-uterine every 8 h did not inhibit luteolysis in heifers, but infusion of estradiol + PGE 2 inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE 1 or PGE 2 prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE 1, or PGE 2 and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE 1 or PGE 2 on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ ( P > 0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13–19 were lower ( P < 0.05) than those treated with intra-luteal implants containing PGE 1 or PGE 2. Day-13 Angus luteal weights were heavier ( P < 0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar ( P > 0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE 1 or PGE 2. Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE 1 or PGE 2 differed ( P < 0.05) from controls, but profiles of progesterone did not differ ( P > 0.05) between breeds or between cows treated with intra-luteal implants containing PGE 1 or PGE 2. Intra-luteal implants containing PGE 1 or PGE 2 prevented ( P < 0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE 1 or PGE 2 alone delays luteolysis regardless of breed. We also conclude that either PGE 1 or PGE 2 prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue.

Androstenedione acts on the coeliac ganglion and modulates luteal function via the superior ovarian nerve in the postpartum rat

Androstenedione can affect luteal function via a neural pathway in the late pregnant rat. Here, we investigate whether androstenedione is capable of opposing to regression of pregnancy corpus luteum that occurs after parturition, indirectly, from the coeliac ganglion. Thus, androstenedione was added into the ganglionar compartment of an ex vivo coeliac ganglion–superior ovarian nerve–ovary system isolated from non-lactating rats on day 4 postpartum. At the end of incubation, we measured the abundance of progesterone, androstenedione and oestradiol released into the ovarian compartment. Luteal mRNA expression and activity of progesterone synthesis and degradation enzymes, 3β-hydroxysteroid-dehydrogenase (3β-HSD) and 20α-hydroxysteroid-dehydrogenase (20α-HSD), respectively, as well as the aromatase, Bcl-2, Bax, Fas and FasL transcript levels, were also determined. Additionally, we measured the ovarian release of norepinephrine, nitric oxide and luteal inducible nitric oxide synthase (iNOS) mRNA expression. The presence of androstenedione in the ganglion compartment significantly increased the release of ovarian progesterone, androstenedione and oestradiol without modifying 3β-HSD and 20α-HSD activities or mRNA expression. The ovarian release of oestradiol in response to the presence of androstenedione in the ganglion compartment declined with time of incubation in accord with a reduction in the aromatase mRNA expression. Androstenedione added to the ganglion compartment decreased FasL mRNA expression, without affecting luteal Bcl-2, Bax and Fas transcript levels; also increased the release of norepinephrine, decreased the release of nitric oxide and increased iNOS mRNA. In summary, on day 4 after parturition, androstenedione can mediate a luteotropic effect acting at the coeliac ganglion and transmitting to the ovary a signaling via a neural pathway in association with increased release of norepinephrine, decreased nitric oxide release, and decreased expression of FasL.

Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cows

The objective of the present study was to investigate if plasma progesterone (pP 4) concentrations are dependent on luteal size, blood flow, or gene expression in luteal tissue. To induce cycles with high and low pP 4 concentrations, respectively, 20 lactating dairy cows received either a single treatment with 25 mg prostaglandin F 2α (PGF 2α) on Day 4 Hour 12 (PG1; n = 8), or two treatments (25 mg PGF 2α each) on Day 4 Hours 0 and 12 (PG2; n = 12) of the estrous cycle (Day 1, Hour 0 = ovulation). In four cows, ovulation occurred between 4 and 6 d after the second PGF 2α treatment; these cows and one lame cow were excluded from the study. In the 15 remaining cows with physiological interovulatory intervals, pP 4, area (LTA) and volume (LTV) of luteal tissue, as well as absolute (LBF) and relative (rLBF) luteal blood flow were determined on Day 9, and relative luteal P 4 (rLP 4) as well as luteal mRNA expression of important receptors, angiogenic, vasoactive, and steroidogenic factors were quantified on Day 11 (±1) during two successive estrous cycles. Furthermore, rLP 4 was multiplied by LTV to produce a semiquantitative assessment of absolute luteal P 4 (LP 4). There was no effect ( P > 0.05) of treatment (one or two PGF 2α treatments), neither on pP 4 concentrations nor on any other parameter in the present study. Nevertheless, there was a lower LP 4 ( P = 0.01), LTA ( P = 0.03), and LTV ( P = 0.02), as well as tendencies of lower pP 4 ( P = 0.06) and LBF ( P = 0.09) at first compared with second diestrus. Plasma P 4 was related with LP 4 ( r = 0.43, P = 0.04), LTA ( r = 0.65, P = 0.0001), and LTV ( r = 0.43, P = 0.02), but not with rLBF ( r = −0.18, P = 0.34). Furthermore, there was no significant correlation between gene expression of important steroidogenic factors and P 4 concentrations in luteal tissue. Results indicate that plasma P 4 concentrations in the mid-luteal phase were dependent on luteal size, but independent of blood flow and gene expression per luteal tissue unit.

Effects of endocannabinoid 1 and 2 (CB1; CB2) receptor agonists on luteal weight, circulating progesterone, luteal mRNA for luteinizing hormone (LH) receptors, and luteal unoccupied and occupied receptors for LH in vivo in ewes

Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F 2α (PGF 2α) starting on days 12–13 post-estrus in ewes with up to 4–6 pulses per day. Prostaglandin F 2α is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F 2α (PGF 2α) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 μg; N = 5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6–48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and occupied LH receptors and mRNA for LH receptors. Profiles of jugular venous progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors were decreased ( P ≤ 0.05) by CB1 or CB2 receptor agonists when compared to Vehicle controls. Progesterone in 80 percent of CB1 or CB2 receptor agonist-treated ewes was decreased ( P ≤ 0.05) below 1 ng/ml by 48 h post-treatment. It is concluded that the stimulation of either CB1 or CB2 receptors in vivo affected negatively luteal progesterone secretion by decreasing luteal mRNA for LH receptors and also decreasing occupied and unoccupied receptors for LH on luteal membranes. The corpus luteum may be an important site for endocannabinoids to decrease fertility as well as negatively affect implantation, since progesterone is required for implantation.

Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF 2α secretion; however, uterine PGF 2α is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF 2α-induced luteolysis, which is via the 2-fold increase in prostaglandins E 1 and E 2 (PGE 1, PGE 2; PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE 1 or PGE 2 prevents spontaneous or an estradiol-17β, IUD, or PGF 2α-induced luteolysis. Four PGE receptor subtypes (EP 1, EP 2, EP 3, and EP 4) and an FP receptor specific for PGF 2α have been identified. The objective of this experiment was to determine the effects of EP 1, EP 2, EP 3, or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE 2 (EP 1, EP 3), butaprost (EP 2), 19-(R)-OH-PGE 2 (EP 2), sulprostone (EP 1, EP 3), or PGF 2α (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE 2 had no effect ( P ≥ 0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE 2 increased ( P ≤ 0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF 2α decreased ( P ≤ 0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP 3 and FP receptors may be involved in luteolysis. In addition, EP 2 receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function.

Prostaglandin E 1 (PGE 1), but not prostaglandin E 2 (PGE 2), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied LH receptors and mRNA for LH receptors in ovine luteal tissue to prevent luteolysis

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF 2α secretion. However, uterine PGF 2α secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF 2α. Prostaglandins E (PGE; PGE 1 + PGE 2) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE 1 or PGE 2 prevents spontaneous or IUD, estradiol-17β, or PGF 2α-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE 1 or PGE 2 affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE 1, or PGE 2 every 4 h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary. Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF 2α and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE 1 or PGE 2 for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE 1 or PGE 2 and day-10 control ewes were similar ( P ≥ 0.05), but were greater ( P ≤ 0.05) than in day-16 Vehicle-treated ewes. Progesterone profiles on days 10–16 differed ( P ≤ 0.05) among treatment groups: PGE 1 > PGE 2 > Vehicle-treated ewes. Concentrations of PGF 2α and PGE in uterine venous plasma on day-16 were similar ( P ≥ 0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar ( P ≥ 0.05) in day-10 control ewes and day-16 ewes treated with PGE 2 and were lower ( P ≤ 0.05) in day-16 Vehicle-treated ewes. PGE 2 prevented loss ( P ≤ 0.05) of day-16 luteal mRNA for LH receptors and occupied and unoccupied LH receptors. Luteal and caruncular tissue mRNA for LH receptors and occupied and unoccupied LH receptors were greater ( P ≤ 0.05) on day-16 of PGE 1-treated ewes than any treatment group. mRNA for LH receptors and occupied and unoccupied receptors for LH in caruncules were greater ( P ≤ 0.05) in day-16 Vehicle or PGE 2-treated ewes than in day-10 control ewes. It is concluded that PGE 1 and PGE 2 share some common mechanisms to prevent luteolysis; however, only PGE 1 increased luteal and endometrial mRNA for LH receptors and occupied and unoccupied LH receptors. PGE 2 prevents a decrease in luteal mRNA for LH receptors and occupied and unoccupied receptors for LH without altering endometrial mRNA for LH receptors or occupied and unoccupied receptors for LH.

Mechanisms of reduced luteal sensitivity to prostaglandin F 2α during maternal recognition of pregnancy in ewes

During maternal recognition of pregnancy, the conceptus stimulates endometrial secretion of PGF 2α and PGE 2. However, PGF 2α is less effective in causing luteal regression in pregnant than in non-pregnant ewes. Experiments were conducted to elucidate mechanisms for reduced luteal sensitivity to PGF 2α during maternal recognition of pregnancy. Corpora lutea (CL) were collected from pregnant and non-pregnant ewes 0, 4, or 12 h following treatment with PGF 2α on day 12 after estrus. Luteal PTGHS2 mRNA did not differ due to PGF 2α or pregnancy status. Luteal PTGES mRNA was reduced in both pregnant and non-pregnant ewes after PGF 2α treatment; while, luteal PTGFS mRNA was reduced 4 h after PGF 2α in pregnant, but not non-pregnant ewes. The result was a greater ratio of PTGES to PTGFS mRNA in pregnant ewes. Luteal mRNA for HPGD did not differ between pregnant and non-pregnant ewes on day 12. Luteal END1 mRNA was reduced in pregnant as compared to non-pregnant ewes prior to PGF 2α challenge. Luteal END1 mRNA was increased after PGF 2α in pregnant and non-pregnant ewes; however, ECE1 mRNA was reduced 4 h after PGF 2α in pregnant, but not non-pregnant ewes. The in vitro conversion of PGF 2α to PGFM was greater in CL of pregnant than non-pregnant ewes at day 14. Luteal conversion of PGF 2α to PGFM appears to be regulated post-transcriptionally. During maternal recognition of pregnancy, mechanisms of reduced luteal sensitivity to PGF 2α may include a shift in prostaglandin production to the luteotropin PGE 2, a reduction of ECE1 mRNA, and increased catabolism of PGF 2α.

Endophyte‐infected tall fescue diet alters gene expression in heifer luteal tissue as revealed by interspecies microarray analysis

Cattle consuming endophyte-infected tall fescue grass have an associated reduction in circulating progesterone and reduced reproductive rates. In this study, commercially available rat microarrays were used to analyze the gene expression in luteal tissues from heifers fed endophyte-free fescue, endophyte-infected fescue, or endophyte-infected fescue supplemented with the dopamine (DA) antagonist, domperidone. The number of hybridized spots represented approximately 40% of the total 10,000 rat genes/ESTs evaluated. Each luteal sample was analyzed in triplicate, resulting in within treatment correlation coefficients of >/=0.98. Median values of mRNA abundance from luteal tissue taken from the endophyte-infected fed heifers revealed 598 genes and ESTs that were down regulated and 56 genes and ESTs that were upregulated compared with luteal mRNA values from the endophyte-free treatment. There were fewer comparative differences between median values from luteal mRNA from the endophyte-free versus feeding endophyte-infected plus domperidone treated heifers. Only 19 genes and ESTs were upregulated and two were down-regulated.