Cellular mechanisms responsible for development of sensitivity of the bovine corpus luteum to prostaglandin F2 alpha

Prostaglandin F2 alpha (PGF2alpha) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF 2alpha is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is an insensitivity of the early CL to luteolytic actions of PGF2alpha. The mechanisms underlying this differential luteal sensitivity are poorly understood. Therefore the main objective of the current study is to understand the cellular mechanism of luteal insensitivity. The developing CL has a maximum number of PGF2alpha receptors; therefore differences in signaling events might be responsible for luteal insensitivity. Hence differential gene expression at two developmental stages of CL, days 4 (D-4) and 10 (D-10) post estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. For example, differential expression of protein kinase C epsilon (PKCepsilon /PRKCE) and its ability to regulate PGF2alpha-stimulated rise in intracellular calcium concentration have been proposed to be part of luteal resistance mechanism. Therefore the current study investigates the: (1) physiological role of PRKCE in regulating the ability of PGF2alpha to inhibit progesterone synthesis, (2) role of PGF2alpha-stimulated rise in intracellular calcium in progesterone inhibitory actions of PGF2alpha, (3) differential expression of a large portion of the luteal transcriptome during its developmental transition from early to mature stage, and (4) role of differentially expressed CAMKK2 in acquisition of luteolytic sensitivity to PGF2alpha . Down-regulation of PRKCE significantly reduced the ability of PGF 2alpha to inhibit LH-stimulated progesterone accumulation. A pharmacological increase in intracellular calcium concentration [Ca2+] i significantly inhibited LH-stimulated progesterone accumulation irrespective of luteal developmental stage. More importantly, buffering the rise in [Ca 2+]i reduced the ability of PGF2alpha to inhibit progesterone accumulation. Microarray analysis identified 167 genes that were expressed differentially (p \u3c 0.05). These were categorized into genes involved in cell signaling (12%), steroidogenesis and metabolism (10.2%), protein degradation (5.3%), transcription regulation and DNA biosynthesis (18.5%), protein biosynthesis and modification (18.5%), extracellular matrix and cytoskeletal proteins (9.5%), antioxidant property (3%), miscellaneous (17%), and unknown functions (6%). In addition, the in vivo administration of PGF2alpha increased the expression of a guanine nucleotide binding protein (G protein), beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin dependent kinase kinase 2, beta (CAMKK2) in D-10 CL. Furthermore, large and small luteal steroidogenic cells, known to be targets for actions of PGF2alpha were demonstrated to be a cellular source for CAMKK2. More importantly, in vitro, a CAMKK2 inhibitor significantly reduced the ability of PGF2alpha to inhibit progesterone accumulation. In summary, a developmental increase in PRKCE expression combined with its ability to regulate [Ca2+]i and the availability of CAMKK2 to mediated the actions of rise in [Ca2+]i might be important components of the mechanism rendering the bovine CL sensitive to PGF2alpha

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM

Effect of the cell-permeable calcium chelator, Bapta-AM, on basal and LH-stimulated progesterone synthesis/secretion (ng/ml) in cultured steroidogenic cells collected Day 10 bovine CL

<p><b>Copyright information:</b></p><p>Taken from "PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells"</p><p>Reproductive biology and endocrinology : RB&E 2007;5():37-37.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2041951.</p><p></p> Progesterone accumulated in culture media was determined after 4 h of incubation in the following treatments: media alone (Media), LH (100 ng/ml), LH and PGFα (1000 ng/ml), or LH and Bapta-AM (0.1, 1, 10, and 100 μmol). As explained in Materials and Methods, these treatments also contained 0.1% of the solvent used for PGFα and Bapta-AM, DMSO. Data are presented as the mean ± SEM of four Day 10 individual replicates (n = 4 CL obtained from 4 cows). Statistical comparisons were made across treatments, and means with different letters denote different values, P < 0.05

Efecto de la administración crónica del antagonista del receptor tipo a (bq-610) de la endotelina 1 sobre el estado funcional del cuerpo lúteo en ovejas

To test the role of endothelin 1 (END1) in luteolysis, an END receptor type A antagonist (BQ-610) was delivered into the corpus luteum (CL) during spontaneous luteolysis in sheep. On day 9 of the estrous cycle, an osmotic mini-pump containing 2 mg of BQ-610 (n = 12) or vehicle (n = 9) were implanted surgically in the ewes. Corpora lutea were collected 12 h after onset of estrus, or on the afternoon of day 21 in ewes that had not returned to estrus, and from untreated ewes on day 10 to 12 of the estrous cycle (mid-cycle CL). Three of 12 BQ-610-treated ewes did not show estrus before day 21 compared to 0 of 9 vehicle-treated ewes. Estrous cycles in vehicle-treated ewes averaged 15.5 ± 0.2 days. In the three BQ-610-treated ewes, luteal weights on day 21 were greater than in vehicle-treated ewes on the day of estrus (0.62 ± 0.05 versus 0.39 ± 0.03 g, respectively; P < 0.001), as were luteal contents of progesterone (P4) (20958.2 ± 1830.9 μg/g versus 1291.2 ± 1057.1 μg/g respectively; P<0.0001). Serum concentrations of P4 in the three BQ-610-treated ewes remained above 1.5 ng/mL through day 21 (P<0.01). Their luteal tissue appeared normal with 53.3 ± 5.8% of apoptotic cells, whereas luteal tissue in vehicletreated ewes was markedly disorganized and in an advanced stage of structural regression. Expression of mRNA of several genes involved in progesterone production or structural luteolysis was different in CL from vehicle-treated compared to CL of BQ-610-treated ewes or mid-cycle CL. In conclusion, chronic infusion of BQ-610 blocked luteolysis and lengthened the estrous cycle in three of 12 ewes. Furthermore, functional features of CL of those three ewes were similar to mid-cycle CL. Overall this study indicates that END1 might plays mediatory role during spontaneous luteolysis in the ewe via endothelin receptor type [email protected] el fin de probar el rol de la endotelina 1 (END1) en la luteólisis, un antagonista del receptor tipo A (BQ-610) de END1 fue administrado dentro del cuerpo lúteo (CL) durante la regresión luteal espontánea en la oveja. En el día 9 del ciclo estrual se implantó quirúrgicamente en el CL de las ovejas una mini bomba osmótica con 2 mg de BQ-610 (n = 12) o un vehículo (n = 9). Los CL fueron colectados 12 horas posteriores al inicio del estro, o en la tarde del día 21 del ciclo estrual en las ovejas que no retornaron en celo, y entre los días 10 a 12 del ciclo estrual en ovejas controles. Tres de 12 ovejas tratadas con BQ-610 (OTBQ-610) no mostraron signos de estro antes del día 21 del ciclo, comparado con 0 de 9 de las que recibieron vehículo (OTV). En las OTV el ciclo estrual promedió 15,5 ± 0,2 días. El peso del CL en las tres OTBQ-610 en el día 21 del ciclo fue mayor que en las OTV en el día del celo (0,62 ± 0,05 versus 0,39 ± 0,03 g, respectivamente; P<0,001); lo cual también ocurrió con el contenido luteal de progesterona (P4) (20958,2 ± 1830,9 μg/g versus 1291,2 ± 1057,1 μg/g respectivamente; P<0,0001). La concentración sérica de P4 en las tres OTBQ-610 permaneció sobre 1,5 ng/mL hasta el día 21 (P<0,01), y el CL tuvo una apariencia normal, con 53,3 ± 5,8% de células apoptóticas; en las OTV el tejido luteal se observó en un estado avanzado de regresión estructural. La expresión del ARNm de algunos genes involucrados en la producción de P4 o en la luteólisis estructural fue diferente en los CL de OTV comparado a los de OTBQ-610 o a los colectados en la fase luteal media. En conclusión, la infusión crónica de BQ-610 dentro del CL bloqueó la luteolisis y prolongó la duración del ciclo estrual en tres de 12 ovejas. En general, el presente estudio indica que END1, a través del receptor tipo A, pudiera ejercer un rol mediador durante la luteolisis espontánea en la oveja

Effect of the Caionophore, A23187, on basal and LH-stimulated progesterone synthesis/secretion (ng/ml) in cultured steroidogenic cells collected from Day 4 (panel A) and Day 10 (panel B) bovine CL

<p><b>Copyright information:</b></p><p>Taken from "PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells"</p><p>Reproductive biology and endocrinology : RB&E 2007;5():37-37.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2041951.</p><p></p> Progesterone accumulated in culture media was determined after 4 h of incubation in the following treatments: media alone (Media), LH (100 ng/ml), LH and PGFα (1000 ng/ml), or LH and A23187 (0.1, 1, 10, and 100 μmol). As explained in Materials and Methods, these treatments also contained 0.1% of the solvent used for PGFα and A23187, DMSO. Data are presented as the mean ± SEM of four Day 4 and 10 Day 10 individual replicates (n = 4 and 10 cows respectively). Statistical comparisons were made across treatments, and means with different letters, differ within each panel (P < 0.05)