Luteal P4 Research Articles

Luteal Expression of Thyroid Hormone Receptors During Gestation and Postpartum in the Rat

Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. TRα1, TRα2, and TRβ1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRβ1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRβ1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

The effect of equine chorionic gonadotropin on follicular size, luteal volume, circulating progesterone concentrations, and pregnancy rates in anestrous beef cows treated with a novel fixed-time artificial insemination protocol

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 μg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm3 and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm3 and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.

Evaluation of treatments with hCG and carprofen at embryo transfer in a demi-embryo and recipient virgin heifer model

An in vivo model, combining a low developmental competence embryo (demi-embryo) and a high-fertility recipient (virgin dairy heifer) was used to evaluate the effects of treatment with human chorionic gonadotropin (hCG) and carprofen at embryo transfer (ET) on plasma progesterone (P4) concentrations of recipients and on embryonic growth and survival. Embryos were bisected and each demi-embryo was transferred to a recipient on Day 7 of the estrous cycle. At ET, heifers (n = 163) were randomly allocated to treatment with hCG (2500 IU im), carprofen (500 mg iv), hCG plus carprofen or to untreated controls. Plasma P4 concentrations were measured on Days 0, 7, 14 and 21 of all recipients plus on Days 28, 42 and 63 of pregnant recipients. Pregnancy was presumed to be present in recipients with luteal plasma P4 concentrations until Day 21 and confirmed by using transrectal ultrasonography on Days 28, 42 and 63. Embryonic measurements (crown–rump length and width) were obtained on Day 42. Treatment with hCG induced formation of secondary corpora lutea (CL) in 97% of heifers and increased (P < 0.01) mean plasma P4 concentrations of non-pregnant recipients on Day 14 and of pregnant heifers on Days 14 to 63. This was associated to a significant decrease in early embryonic mortality. In contrast, subsequent embryonic losses resulted in a non-significant numerical increase by 8% of pregnancies maintained to Day 63. Therefore, treatment with hCG significantly rescued embryos through the maternal recognition of pregnancy window but was not able to support development thereafter. Treatment with carprofen at ET had no significant effects on plasma P4 concentrations and rate of embryo mortality. Treatment with hCG plus carprofen at ET induced formation of secondary CL in 90% of heifers but decreased the luteotrophic effect of hCG, resulting in no effect on embryo survival. Low developmental competence embryos showed an intrinsic deficiency in overcoming the maternal recognition of pregnancy challenge and in proceeding to further development until Day 28 of pregnancy, whereas mortality beyond this point was residual. Results on pregnancy rates should be confirmed in further experiments involving a larger sample size.

Luteoprotective Mechanisms of Prostaglandin F2α Stimulated by Luteinizing Hormone in the Bovine Corpus Luteum

Luteinizing hormone (LH) regulates several ovarian functions. However, the luteoprotective mechanisms of LH involved in the maintenance of bovine corpus luteum (CL) function are not well understood. Since prostaglandin F2α (PGF), PGE2 and progesterone (P4) are well documented as antiapoptotic factors in the bovine CL, we hypothesized that LH protects the CL by stimulating the local production and action of PGF, PGE2 and P4. Cultured bovine luteal cells obtained at the mid-luteal stage (days 8–12 of the estrous cycle) were treated with LH (10 ng/ml), onapristone (OP: a specific P4 receptor antagonist, 100 μM) and indomethacin [INDO; a cyclooxygenase (COX) inhibitor, 100 μM] for 24 h. LH with and without OP significantly increased the mRNA and protein expressions of COX-2, PGF synthase and carbonyl reductase (P<0.05) but not the mRNA and protein expressions of COX-1 and PGE synthase in bovine luteal cells. In addition, these treatments significantly increased PGF and P4 production (P<0.05) but not PGE2 production. Luteal cell viability was significantly increased by LH alone (P<0.05), but LH-increased cell viability was reduced by LH in combination with INDO as well as OP (P<0.05). The overall results suggest that LH prevents luteal cell death by stimulating luteal PGF and P4 production and supports CL function during the luteal phase in cattle.

Plasma progesterone concentration in beef heifers receiving exogenous glucose, insulin, or bovine somatotropin1

Three experiments were conducted to evaluate plasma concentrations of glucose, insulin, IGF-I, and progesterone (P4) in pubertal beef heifers receiving exogenous glucose, insulin, or sometribove zinc. All heifers used had no luteal P4 synthesis but received a controlled internal drug-releasing device containing 1.38 g of P4 to estimate treatment effects on hepatic P4 degradation. In Exp. 1, 8 pubertal, nulliparous Angus × Hereford heifers (initial BW = 442 ± 14 kg; initial age = 656 ± 7 d) were randomly assigned to receive, in a crossover design containing 2 periods of 10 h, intravenous (i.v.) infusions (10 mL) of insulin (1 μg/kg of BW; INS) or saline (0.9%; SAL). Treatments were administered via jugular venipuncture in 7 applications (0.15 μg insulin/kg BW per application) 45 min apart (from 0 to 270 min). Blood samples were collected immediately before each infusion as well as at -120, -60, 330, 390, and 450 min relative to the first infusion. Heifers receiving INS had greater (P < 0.01) plasma insulin, reduced (P ≤ 0.04) plasma glucose and IGF-I, and similar (P = 0.62) plasma P4 concentrations compared with SAL heifers. In Exp. 2, the same heifers were assigned to receive, in a similar experimental design as Exp. 1, i.v. infusions (10 mL) of 1) insulin (1 μg/kg BW) and glucose (0.5 g/kg BW; INS+G) or 2) SAL. Heifers receiving INS+G had greater (P ≤ 0.02) plasma insulin, glucose, and P4 but reduced (P = 0.01) plasma IGF-I concentrations compared with SAL heifers. In Exp. 3, the same heifers were assigned to receive, in a crossover design containing 2 periods of 14 d, subcutaneous (s.c.) injections of 1) 250 mg of sometribove zinc (BST) or 2) SAL. Blood samples were collected 3 h apart (0900, 1200, 1500, and 1800 h) from heifers on d 6, 8, and 10 relative to treatment administration (d 1). Heifers receiving BST had greater (P < 0.01) plasma glucose and IGF-I and similar (P ≥ 0.67) plasma insulin and P4 concentrations compared with SAL heifers. Results from this series of experiments suggested that concurrent increases in glucose and insulin are required to reduce hepatic catabolism and increase plasma concentrations of P4 in bovine females.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity and protein expression

Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1–2 days after ovulation), middle (CL2: 7–10 after ovulation), and late luteal phase (CL3: 13–15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Effect of oxytocin infusion on luteal blood flow and progesterone secretion in dairy cattle

The objective of this study was to investigate the effects of oxytocin infusion on corpus luteum (CL) function during early to mid-diestrus by measuring luteal size (LS) and luteal blood flow (LBF) along with plasma levels of progesterone (P4) and prostaglandin metabolites (13,14-dihydro-15-keto-prostaglandin F2α, PGFM). On day (D) 7 of the estrus cycle (D1 = ovulation), seven cows received 100 IU of oxytocin (OXY) or placebo (PL) following a Latin square design. LS and LBF increased in both groups over time and no differences were observed between the groups. PGFM did not differ either within the groups over time or between the groups at any time point. P4 of the OXY group was higher compared to that of the the PL group 360 min after the infusion (p = 0.01) and tended to be higher at the time points 450 min, 48 h, and 72 h (all p = 0.08). Results from this study support the hypothesis that OXY is not directly involved in the mechanism(s) governing blood flow of the CL and has no remarkable effects either on luteal size or P4 and PGFM plasma levels. Further investigation is needed to elucidate the role of OXY in CL blood flow during early and late luteal phases.

The influence of steroids on noradrenaline-mediated contractile reactivity of the superficial nasal and facial veins in cycling gilts

The nasal venous blood may be directed through the facial vein into the systemic circulation or through the frontal vein into the venous cavernous sinus of the perihypophyseal vascular complex, where hormones and pheromones permeate from the venous blood into the arterial blood supplying the brain and hypophysis. The present study was designed to determine the effect of noradrenaline (NA) on the tension of the nasal, frontal and facial veins of cycling gilts, and influence of ovarian steroid hormones on NA-mediated contractile reactivity. Additionally, the enzyme dopamine-beta-hydroxylase catalysing the conversion of dopamine to noradrenaline (DbetaH) was immunolocalized in these vessels. Among three studied veins, the frontal proximal vein, that fulfill a key role in the supply of the nasal venous blood into the venous cavernous sinus, reacted to NA most strongly (P < 0.001) and this reaction was weaker in the periestrous period than in luteal phase (P < 0.001). Inversely, the reaction to NA of the facial proximal vein, that carry blood to the peripheral circulation, was stronger in the periestrous period than in luteal phase (P < 0.05). P4, E2 and T significantly lowered NA-mediated tension of the frontal proximal vein during the periestrous period (P < 0.001), while in the luteal phase P4 might antagonize relaxing effect of E2 to this vessel. The result suggests that supply of the nasal venous blood into the venous cavernous sinus is greater during the periestrous period than during the luteal phase. DbetaH was clearly expressed in the muscular layer of the isolated superficial nasal and facial veins of gilts in both studied stages of the estrous cycle. We suggest that the reactivity of the superficial veins of the nose and face to NA combined with the previously demonstrated reactivity of these veins to steroid ovarian hormones and male steroid pheromones may regulate the access of priming pheromone androstenol (resorebed in the nasal cavity) to the brain of gilts during periestrous period via humoral local destination transfer.

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P₄) levels tend to change with the season, little is known about the seasonal changes of P₄ synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P₄ concentration, was used to evaluate the seasonal changes in P₄ synthesis. The luteal P₄ concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P₄ synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P₄ synthesis-related proteins in mares.

Differential Stimulation Pathways of Progesterone Secretion from Newly Formed Corpora Lutea in Rats Treated with Ethylene Glycol Monomethyl Ether, Sulpiride, or Atrazine

Ethylene glycol monomethyl ether (EGME), sulpiride, and atrazine are known ovarian toxicants, which increase progesterone (P4) secretion and induce luteal cell hypertrophy following repeated administration. The aim of this study was to define the pathways by which these compounds exerted their effects on the ovary and hypothalamic-pituitary-gonadal (HPG) axis. In the ovary, changes in the steroidogenic activity of new and old corpora lutea (CL) were addressed. EGME (300 mg/kg), sulpiride (100 mg/kg), or atrazine (300 mg/kg) were orally given daily for four times from proestrus to diestrus in normal cycling rats. Treatment with all chemicals significantly increased serum P4 levels, and EGME as well as sulpiride induced increases in prolactin (PRL) levels. In new CL, at both the gene and the protein levels, all three chemicals upregulated the following steroidogenic factors: scavenger receptor class B type I, steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, and 3β-hydroxysteroid dehydrogenase (HSD) and downregulated the luteolytic gene, 20α-HSD. Coadministration of EGME and bromocriptine, a D2 agonist, completely inhibited PRL but not P4 secretion. Additionally, steroidogenic factor expression levels were upregulated, and 20α-HSD level was downregulated in new CL. These results suggest that EGME both directly and indirectly stimulates P4 production in luteal cells, whereas sulpiride elevates P4 through activation of PRL secretion in the pituitary. Atrazine may directly activate new CL by stimulating steroidogenic factor expressions. The present study suggests that multiple pathways mediate the effects of EGME, sulpiride, and atrazine on the HPG axis and luteal P4 production in female rats in vivo.

Luteal and placental function in the bitch: spatio-temporal changes in prolactin receptor (PRLr) expression at dioestrus, pregnancy and normal and induced parturition

BackgroundEndocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog.MethodsThe expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization.ResultsIn non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated.ConclusionsThe data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.

Subfertility Linked to Combined Luteal Insufficiency and Uterine Progesterone Resistance

Early pregnancy loss is common and can be caused by a range of factors. The Brown Norway (BN) rat exhibits reproductive dysfunction characterized by small litter size and pregnancy failure and represents a model for investigating early pregnancy loss. In this study, we investigated the establishment of pregnancy in the BN rat and gained insight into mechanisms causing its subfertility. Early stages of BN uteroplacental organization are unique. The BN primordial placenta is restricted in its development and correlates with limited BN uterine decidual development. BN uterine decidua was shown to be both structurally and functionally distinct and correlated with decreased circulating progesterone (P4) levels. Ovarian anomalies were also apparent in BN rats and included decreased ovulation rates and decreased transcript levels for some steroidogenic enzymes. Attempts to rescue the BN uterine decidual phenotype with steroid hormone therapy were ineffective. BN uteri were shown to exhibit reduced responsiveness to P4 but not to 17beta-estradiol. P4 resistance was associated with decreased transcript levels for the P4 receptor (Pgr), a P4 receptor chaperone (Fkbp4), and P4 receptor coactivators (Ncoa1 and Ncoa2). In summary, the BN rat exhibits luteal insufficiency and uterine P4 resistance, which profoundly affects its ability to reproduce.

Effects of Atosiban on Progesterone Production During Early and Mid Cycle in the Ewe.

The role of oxytocin (OT) in the ovine CL is not fully characterized. Oxytocin has displayed either luteolytic or luteotrophic effects depending on the protocol used and mode of administration. Oxytocin has been proposed to be a mediator of PGF2alpha-induced luteolysis. An experiment was designed to test the role of OT in PGF2alpha-induced luteolysis. Mid-ventral laparotomy was performed between days 7.5 to 9.0 of the estrous cycle (d 0 = estrus). To prepare ewes for surgery, 0.14 mg/kg of Ketamine (Columbus Serum, Columbus, OH; 100 mg/mL) and 0.3 mg/kg diazepam (Columbus Serum, Columbus, OH; 5 mg/mL) were injected i.v. and isoflurane (Columbus Serum, Columbus, OH) was used to maintain anesthesia during surgery. An osmotic mini-pump was sutured into the ovarian pedicle near the hilus of the ovary, and the catheter was inserted directly into the CL. In animals with multiple CL, one ovary was removed, or one CL was enucleated so as to leave only one CL. The osmotic mini-pump contained an OT receptor antagonist, Atosiban (ATO; Sigma, St. Louis, MO), in dilutions to deliver 0.25 (n = 3), 1.25 (n = 2), 2.5 (n = 3), or 5.0 (n = 3) µg/h or vehicle (70% Methanol: 30% Saline) in 0.5 µL/h. Forty-eight hours after surgery, 25 mg of PGF2alpha (Lutalyse Pfizer Animal Health, NY, NY) was injected i.m. (h = 0). Jugular blood samples (10 mL) were taken every 3 h beginning at h 0 and continued for 24 hours. At 24 h, ewes were euthanized, attachment of the catheter was verified, and the ovary was collected. Serum P4 in ewes treated with 2.5 or 5.0 µg/h ATO was lower than in vehicle-treated or control ewes at 0 h (P < 0.13 and P < 0.05 respectively). Content of P4 in CL was lower in PGF2alpha-treated than in control ewes (P < 0.04). Due to unexpected negative effects of ATO on P4 concentration, a second experiment was designed to determine if P4 concentration was reduced by ATO earlier in the estrous cycle, when the CL is insensitive to a single treatment with PGF2alpha. Osmotic mini-pumps delivering either 5.0 μg/h ATO (n = 4) or vehicle (n = 5) were implanted via mid-ventral laparotomy on day 3 of the estrous cycle. The same protocol as in experiment 1 was followed except that blood samples were collected every 8 h for 48 h after pump implant, and ATO was the only treatment. The pattern of mean serum concentrations of P4 during hours 16 through 48 was lower (P < 0.002) in ATO-treated than in vehicle-treated ewes (1.37, 1.04, 0.68, 0.84, 0.78, 0.67, and 0.8 ng/mL compared to 1.65, 0.95, 1.35, 1.18, 1.26, 1.36, and 1.56 ng/mL at 0, 8, 16, 24, 32, 40, and 48 h, respectively). Luteal weight and content of P4 in CL did not differ (P = 0.59 and P = 0.22, respectively). In conclusion, ATO (5.0 µg/h) decreased serum P4 concentration in ewes either during early or mid-cycle. Though P4 content in the CL was not decreased significantly (157.1 and 191.8 µg/g for treated and control ewes, respectively), more animals are needed to determine if luteal P4 was reduced. It is suggested that ATO reduced luteal function, perhaps by binding to an OT receptor type that ATO has been shown to stimulate in human myometrium. (poster)

Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2α-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2α, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2α synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid-dehydrogenase (3βHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches ( n = 3–5) on days 8–12 (Group 1, pre-implantation period), days 18–25 (Group 2, post-implantation period), days 35–40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3βHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged. These data suggest that – as in the nonpregnant bitch – also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3βHSD.

An hCG Antagonist Reduces Ovulation and Diminishes OHSS in a Rodent Model

An hCG Antagonist Reduces Ovulation and Diminishes OHSS in a Rodent Model

The Effects of Prolactin and Gonadotropin on Luteal Function and Morphology in the Cyclic Golden Hamster

The present study was conducted to evaluate the endocrinological effects of the pituitary on luteal maintenance and regression in the cyclic golden hamster (Mesocritus auratus). After hypophysectomy (Hypox) at 0900 h on day 1 of the estrous cycle (the day of ovulation), the animals received injection of prolactin (PRL) or PRL plus equine chorionic gonadotropin (eCG). They were decapitated at 1500 h on day 3 of the cycle, and trunk blood was collected for measurement of progesterone (P4). Corpora lutea (CLs) were dissected from one ovary for DNA ladder detection by electrophoresis, determination of DNA fragmentation ratio by fluorometric measurement method and measurement of P4. The other ovary was used for histological observation. After the Hypox, the daily injection of 1 mg ovine PRL restrained the DNA fragmentation ratio and number of apoptotic cell in the CLs. The PRL treatment maintained the luteal morphology and increased the luteal P4 concentration, but not in the plasma P4 concentration. In addition to PRL, injection of 2 IU eCG after the Hypox also restrained the DNA fragmentation ratio and number of apoptotic cells in the CLs to the level of a pregnant animal. The PRL plus eCG treatment maintained the luteal morphology in the same manner as the PRL only treatment and increased not only the luteal but also the plasma P4 concentration. These results suggest that PRL restrains luteal apoptosis and maintains luteal morphology and that the combination of PRL and eCG restrains not only structural but also functional luteal regression in the cyclic hamster.

An hCG antagonist reduces ovulation and diminishes OHSS in a rat model through down-regulation of VEGF

OHSS is a complication of IVF, attributable to changes following hCG treatment to induce final oocyte maturation. These changes increase vascular endothelial growth factor (VEGF) resulting in increased vascular permeability (VP). We have shown that a modified hCG (hCG-Ant) binds competitively to the LH receptor, blocking LH activity in vitro. In a rat OHSS model, hCG-Ant reduces ovulation and significantly diminishes OHSS by reducing VP. We quantified the effect of hCG-Ant on ovulation and OHSS by measuring hormone levels and determining VEGF and VEGF receptor 2 (VEGFR-2) protein localization and mRNA expression in tissue, as well as changes in ovarian weight between treatment groups. In vivo studies used standard rat models for ovulation and OHSS. For ovulation studies, immature female Wistar rats were randomized to 2 groups. 11 rats received 10 IU pregnant mare serum gonadotropin (PMSG) ip for 1d, followed by 10 IU hCG or hCG-Ant 48h later. Rats were sacrificed 24h post-hCG and serum was obtained for E2, P4, and inhibin assays. For OHSS studies, 12 rats were randomized to receive 10 IU PMSG for 4d, followed by 0.1 mL saline (control, n = 4), 30 IU hCG to induce OHSS (n = 4), or 30 IU hCG followed by 30 IU hCG-Ant 24 h later (treatment group, n = 4). After 48 h, tissue was prepared for Western blotting and RT-PCR using standard methods. Ovarian weight was higher in the hCG group vs. hCG-Ant (0.0361 ± 0.014 g vs. 0.019 ± 0.007 g, P<.05). E2 and P4 levels were higher in the hCG vs. hCG-Ant group (91.45 ± 30.84 pg/mL and 6.48 ± 4.13 ng/mL vs. 53.12 ± 18.55 pg/mL and 3.43 ± 2.03 ng/mL, P<.05). Inhibin A increased linearly as luteal E2 and P4 rose. Average inhibin A was higher in the hCG vs. hCG-Ant group (553.63 ± 107.18 pg/mL vs. 247.2 ± 74.19 pg/mL, P<.05). Western blot showed VEGF was present in ovaries treated with hCG and saline and absent in ovaries treated with hCG-Ant following hCG. VEGFR-2 was present in the ovary by Western blot for all groups. RT-PCR confirmed increased ovarian mRNA expression of VEGF in the hCG and saline groups vs. hCG-Ant following hCG. HCG-Ant linearly decreases luteal E2, P4, and inhibin A, reflecting a reduction in ovulation, suggesting a dose-dependent effect. Controlling ovulation may diminish OHSS risk. Ovarian VEGF expression is abolished by giving hCG-Ant after hCG in an OHSS model. Clinically, during an IVF cycle, hCG-Ant may be given following hCG treatment and oocyte retrieval to reduce OHSS while preserving IVF cycle outcome.

Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação

O presente trabalho foi desenvolvido para testar a hipótese de que células luteínicas bovinas em cultivo, provenientes dos três terços de gestação, comportam-se da mesma maneira que células in vivo em relação à produção de P4. Foram coletadas amostras de corpos lúteos (CL) de 90 (n=3), 150 (n=3) e 210 (n=3) dias de gestação obtidos em abatedouro. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 poços. Após 24 horas de cultivo foram feitas a lavagem dos poços e a adição do precursor pregnenolona. Os tratamentos foram realizados em octuplicata para cada tempo de tratamento (24, 48 e 96 horas) com três repetições de cada período gestacional. As amostras de meio de cultura e as células foram coletadas 24, 48 e 96 horas após adição do precursor e acondicionadas em freezer a -20ºC até o processamento. A progesterona foi dosada através de radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os resultados foram analisados estatisticamente e considerados diferentes quando p&lt;0.05. Foi observada maior produção de P4 aos 90 dias de gestação (35,277±0,075), posterior decréscimo aos 150 dias (28,820±0,231) e novo aumento aos 210 dias (32,777±0,099). A produção de P4 em células cultivadas por 24 horas foi maior (p&lt;0,05) em células oriundas do grupo de 90 dias (2,912±0,047) quando comparado a 150 (2,669±0,137) e 210 dias (2,741±0,088). As 48 e 96 horas de cultivo, células luteínicas bovinas de 90 dias produziram mais P4 que células de 210 dias (2,934±0,029 e 2,976±0,121 respectivamente x 2,760±0,059 e 2,695±0,149, respectivamente; p&lt;0,05), que por sua vez produziram mais do que células de 150 dias (2,334±0,084 para 48 horas e 2,205±0,136 para 96 horas). Aos 150 dias de gestação a produção de progesterona apresentou diminuição gradativa ao longo das 96 horas de cultivo. Essas diferenças podem ser explicadas pela expressão gênica diferencial de enzimas ou também de fatores presentes na cascata esteroidogênica de acordo com a idade gestacional. Este modelo de cultura celular luteínica poderá ser utilizado em estudos funcionais uma vez que o padrão de secreção de P4 mimetizou o que ocorre in vivo.

Luteotrophic effect, growth and survival of whole versus half embryos and, their relationship with plasma progesterone concentrations of recipient dairy heifers

This prospective and randomised experiment was designed to compare the luteotrophic effect of whole versus half embryos and, to evaluate the relationship between the plasma progesterone (P4) profiles and the rates of early embryonic (from Days 7 to 25), late embryonic (Days 25–42) and foetal (Days 42–63) mortalities of whole and half embryo recipients. Within a single herd, 188 virgin, healthy, cyclic, reproductively sound, with adequate body condition score, Holstein dairy heifers were randomly allocated to receive one whole or one half embryo on Day 7 of the oestrous cycle (Day 0 = estrus). In each embryo-transfer (ET) group, half of the recipients were treated with a CIDR (controlled internal drug releasing device) between Days 7 and 19. Pregnancy was evaluated by ultrasound on Days 25, 42 and 63 and plasma P4 profiles were obtained until Day 63 of pregnancy. CIDR-treated and untreated heifers had similar pregnancy rates on Days 25, 42 and 63 and, embryo size on Day 42 was also similar in treated and untreated recipients. Therefore, CIDR treatment failed to promote growth and survival of half and whole embryos. Half embryos presented a significantly higher rate of early and late embryonic mortality than whole embryos. In contrast, foetal mortality was similar in whole and half embryos and, this was coincidental to a similar embryo size on Day 42. Therefore, half embryos exhibited a compensatory growth until Day 42, irrespective of CIDR treatment, after which they presented a similar survival rate to that of whole embryos. Half embryo-derived pregnancies presented significantly lower plasma P4 concentrations on Day 25 than whole embryo-derived pregnancies, suggesting that this lower luteotrophic effect of half embryos could be related to their higher rate of late embryonic mortality. No significant relationship between the early luteal P4 concentrations and embryo survival was observed in whole and half embryo recipients. The first detectable luteotrophic effect of embryonic origin was observed on Day 14 and no detectable second luteotrophic effect was observed until Day 63 of pregnancy. Treatment with CIDR significantly increased plasma P4 concentrations during treatment but induced a significant decrease after removal of the device, suggesting that secretion of luteotropins was downregulated in the course of treatment.