Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Impact of castration with or without alpha-tocopherol supplementation on the urethral sphincter of rats

OBJECTIVE: To analyze the impact of low levels of testosterone induced by orchiectomy and the effect of alpha-tocopherol supplementation on oxidative stress in the urethral sphincter. MATERIALS AND METHODS: Forty male Wistar rats weighing 250-300g were divided into four groups with 10 each: Sham group; Orchiectomy group: bilateral orchiectomy; Orchiectomy-pre-Tocopherol group: bilateral orchiectomy preceded by alpha-tocopherol supplementation for four weeks; Orchiectomy-full-Tocopherol group: bilateral orchiectomy with alpha-tocopherol supplementation for four weeks preceding the procedure and for eight weeks afterwards. At the protocol end, animals were euthanized and had the sphincter analyzed stereologically focusing on collagen and muscle fibers percentage. Oxidative stress levels were determined using 8-epi-PGF2. RESULTS: The 8-epi-PGF2 levels were statistically higher (p < 0.0003) in the Orchiectomy group compared to others groups while Sham and Orchiectomy-full-Tocopherol groups presented statistically similar values (p = 0.52). Collagen volumetric densities were significantly lower in Sham and Orchiectomy-full-Tocopherol groups (p < 0.022). Sham group presented statistically greater muscle fiber percent. CONCLUSION: Castration caused oxidative stress in the urethral sphincter complex, with increased collagen deposition. Alpha-tocopherol had a protective effect and its supplementation for twelve weeks provided the greatest protection.

Improving the reproductive efficiency of the goat: vaginal cytology and vulvar biometry as predictors of synchronized estrus/breeding time in West African dwarf goat / Mejora de la eficiencia reproductiva de la cabra: citología vaginal y biometría vulvar como predictores de la sincronización del periodo estro/crianza de cabras enanas del Oeste Africano

The characteristics of exfoliated vaginal cells and vulvar biometry following estrus synchronization via two injections of 5mg Lutalyse® administered 7 days apart were investigated with the aim of their possible use to predict estrus in six adult WAD does. Four adult WAD bucks recently passed as satisfactory potential breeders were also involved in the study. The animals were maintained on 12 percent crude protein concentrate, greens and fresh water ad libitum. All measurements in the does were taken at an interval of 24 hours for six days beginning with the day of 2nd Lutalyse® injection. The does were introduced to the bucks 48 hours after the 2nd dose of Lutalyse® and separated from them after the 6th day. The 72-96 and 96-120 hours vaginal smears of 5 does (i.e. 83.3 percent) were characteristic during the study. They were positive for sperm cells and showed sharp increase in the degree of clumping of exfoliated cells. During these periods also, the differences in the percentage of superficial cells (i.e. 77.4 +/- 1.05 and 56.4 +/- 0.77) over other epithelial cells (12.2 +/- 0.38 and 1.30 +/- 0.82) respectively were significant (P<0.05). The percentage leucocytes also varied during the study but increased sharply during 96 -120 hours. The result on vulvar biometry between 0-72 hours and the period during which mating occurred (i.e. 72-120 hours) was not significant (P>0.05). All does with vaginal smear positive for sperm cells were confirmed pregnant at day 60 following mating by ultrasonography. The results of this study show that two injections of 5mg Lutalyse® 7 days apart will produce fertile estrus in the WAD doe. In conclusion, a careful evaluation of 24 hourly exfoliated vaginal cells will enhance synchronized estrus detection in WAD goat and improve their reproductive efficiency.

Fueron investigadas en seis cabras WAD hembras adultas, las características de las células vaginales exfoliadas y la biometría vulvar, tras una sincronización de estros a través de dos inyecciones de 5 mg de Lutalyse ®, administrados cada 7 días, con el fin de hacer posible el uso y predecir el estro. También participaron en este estudio cuatro machos adultos WAD probados recientemente como potenciales reproductores. Los animales fueron alimentados con un concentrado de proteína cruda de 12 por ciento, pastos y agua ad libitum. Las medidas en las cabras fueron tomadas con un intervalo de 24 horas, durante 6 días, a contar de la segunda inyección Lutalyse ®. Después de 48 horas de la 2 dosis de Lutalyse ® las cabras fueron cruzadas, y separadas de los machos después del 6 día. Fue realizado el estudio en frotis vaginales de 72-96 horas y de 96-120 horas, en 5 hembras (83,3 por ciento). Estos frotis fueron positivos para las células espermáticas y mostraron fuerte aumento en el grado de aglutinación de células de descamación. Durante estos períodos, las diferencias en el porcentaje de células superficiales (77,4 +/- 1,05 y 56,4 +/- 0,77) sobre las células epiteliales de otros (12,2 +/- 0,38 y 1,30 +/- 0,82) fueron significativas (P <0,05). También varió el porcentaje de leucocitos durante el estudio, pero aumentó considerablemente durante el periodo de 96-120 horas. El resultado de biometría vulvar entre 0-72 horas y del período durante el cual se produjo el apareamiento (72-120 horas) no fue significativa (P> 0,05). Todas las hembras con frotis vaginal positivo para células espermáticas, se les confirmó la preñez por ecografía, a los 60 días posterior al apareamiento. Los resultados de este estudio mostraron que dos inyecciones de 5 mg Lutalyse ® con 7 días de diferencia produce estro fértil en la cabra WAD. En conclusión, una evaluación cuidadosa de las células vaginales exfoliadas a las 24 horas, mejorará la detección del estro en cabras WAD y su eficiencia...

Calcium metabolism in cows receiving an intramuscular injection of 1,25-dihydroxyvitamin D3 combined with prostaglandin F2alpha closely before parturition

To determine the effect of exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] combined with induced parturition on calcium (Ca) metabolism, cows received a single intramuscular injection of 1,25(OH)2D3 and prostaglandin F2alpha (PGF2alpha) closely before calving. Ten late-pregnant, multiparous Holstein cows were assigned to 1,25(OH)2D3 group (five treated with both 1,25(OH)2D3 and PGF2alpha) and control group (five treated with PGF2alpha). 1,25(OH)2D3 group showed an increase in plasma Ca concentration around parturition, whereas control group revealed a decrease in plasma Ca level. Plasma Ca concentration in 1,25(OH)2D3 group were significantly higher than that in control group during .0.5 to 3 days after parturition.

Evaluation of tocolytic efficacy of selective beta2 adrenoceptor agonists on buffalo uterus.

Present study was conducted on prostaglandin F2alpha (PGF2alpha), oxytocin, (OT), potassium chloride (KCI) and barium chloride (BaCl2) pre-contracted perimetrial uterine strips of dioestrus and pregnant buffaloes to evaluate the tocolytic efficacy of selective beta2 adrenoceptor agonists-albuterol (salbutamol) and terbutaline. Cumulative concentration-response curves of both the beta2 adrenoceptor agonists were constructed and the mean effective concentration (EC50) values determined and compared statistically. Based on the comparative EC50 values in relaxing the pre-contracted uterine strips with different spasmogens, the rank order potency of albuterol was found to be--PGF2alpha > BaCl2 > OT > KCl on uterine strips from dioestrus animals, while OT> BaCl2> PGF2alpha >KCl on the uterine strips of pregnant buffaloes. The rank order potency of terbutaline on uterine strips from dioestrus stage animals was- BaCl2 > OT > KCl > PGF2alpha, while BaCl2 > PGF2alpha > KCl > OT on uterine tissues of pregnant animals. Thus, irrespective of the state of uterus, whether gravid or non-gravid, KCl-depolarized uterine tissues required comparatively higher concentrations of albuterol or terbutaline to produce tocolytic effect. High concentrations of K+ in biophase may have interfered with the beta2 adrenoceptor agonists-induced outward K+ current and hyperpolarization. From the results of present study, it was evident that selective beta2 adrenergic agonists had good tocolytic efficacy on the uterus of buffaloes. Further, indirectly the possibility of existence and activation of K(Ca) channels by selective beta2 adrenoceptor agonists in mediating tocolysis of buffalo myometrium can not be ruled out, however, detailed studies using specific K(Ca) channel blockers are required for characterizing the nature of such channels in buffalo uterus.

Synchronization of ovulation in crossbred dairy heifers using gonadotrophin-releasing hormone agonist, prostaglandin F2alpha and human chorionic gonadotrophin or estradiol benzoate

Girolando (Gir x Holstein) is a very common dairy breed in Brazil because it combines the rusticity of Gir (Bos indicus) with the high milk yield of Holstein (Bos taurus). The ovarian follicular dynamics and hormonal treatments for synchronization of ovulation and timed artificial insemination were studied in Girolando heifers. The injection of a gonadotrophin-releasing hormone (GnRH) agonist was followed 6 or 7 days (d) later by prostaglandin F2 alpha (PGF2alpha). Twenty-four hours after PGF2alpha injection either human chorionic gonadotropin (hCG, GPh-d6 and GPh-d7 groups) or estradiol benzoate (EB, GPE-d6 and GPE-d7 groups) was administered to synchronize ovulation and consequently allow timed artificial insemination (AI) 24 and 30 h after hCG and EB injection, respectively. Follicular dynamics in Girolando heifers was characterized by the predominance of three follicular waves (71.4 percent) with sizes of dominant follicles (10-13 mm) and corpus luteum (approximately 20 mm) similar to those for Bos indicus cattle. In the GnRH-PGF-hCG protocol, hCG administration induced earlier ovulation (67.4 h, P<0.01) compared to the control group (GnRH-PGF) and a better synchronization of ovulation, since most of it occurred within a period of 12 to 17 h. Pregnancy rate after timed AI was 42.8 (3/7, GPh-d6) to 50 percent (7/14, GPh-d7). In contrast, estradiol benzoate (GnRH-PGF-EB protocol) synchronized ovulation of only 5 of 11 heifers from the GPE-d7 group and of none (0/7) from the GPE-d6 group, which led to low pregnancy rates after timed AI (27.3 and 0 percent, respectively). However, since a small number of Girolando heifers was used to determine pregnancy rates in the present study, pregnancy rates should be confirmed with a larger number of animals

Comparative study on the efficacy of prostaglandin F[2] and short-term norgestomet treatments as luteolytic agents in buffaloes

Induction of luteolysis in suboesterous buffaloes with palpable and functional cor pora lutea was applied. Different doses of prostaglandin F[2]alpha [lutalyse] were used through intramuscular [25 mg] and vaginal submucosa [12.5 and 7.5 mg] routes. Short-term [9-10 days] norgestomet [Synchro-Met-B] ear implants were also administered for comparison. The results showed that intramuscular injection of 25 mg PGF[2] alpha during summer inducd luteolysis in 70% of the treated animals, while injection of 12.5 mg PGF[2]alpha into vaginal submucosa of suboestrous buffaloes during summer also, resulted in luteolysis in 87.5%. Injection of 7.5 mg PGF[2] alpha into vaginal submucosa in suboestrus buffaloes during winter improved the rsponse [88.89%]. Application of Synchro-Met-B ear implants led to luteolysis in 90.90% of the treated buffaloes. In conclusion, injection of PGF[2] alpha into submucosa proved to be effective, economic and easily applied treatment of suboestrous and/or synchronization of oestrus in buffaloes

Actividad Biológica de la Prostaglandina F2 Alfa y del Metil Carboprost en el Fundus Gástrico aislado de rata / Biological activity of prostaglandin Fs alpha and methyl carboprost in the gastric fundus isolated from rats

Se comparó la potencia contráctil de una muestra del éster metílico de 15 metil prostaglandina F2 alfa (metil carboprost) (1), producida por la Industria Farmacéutica Cubana, con una muestra comercial de prostaglandina F2 alfa (II), de la firma Chinoin, mediante el ensayo de los "tres puntos". También se comparó la duración de la contracción inducida por dichas muestras utilizando un análisis de regresión lineal de la relajación observada en función del tiempo, luego de suprimir el contacto entre la droga y el órgano efector. Se utilizó el fundus gástrico aislado de rata como órgano efectos. La actividad contráctil se registró mediante transductores fuerza-desplazamiento. La muestra I presentó una potencia relativa de 164,0 por ciento con respecto a la II, con límites de confianza de 111,2 y 247,8 por ciento (p =0,95; N=7), y mostró un efecto más prolongado en relación con la II. En las tiras contraídas por la muestra I debió transcurrir 6 min y 46 s, luego del lavado, para que la contracción se redujera a la mitad de la respuesta máxima observada (N=7), en tanto que en las tiras contraídas por la II fue de 1 min y 48 s (N=8)

Cardioplegia cristalóide, barotrauma e funçäo endotelial: consideraçöes experimentais / Crystalloid cardioplegia, barotrauma and endothelium function: experimental considerations

O presente ensaio experimental estudou o efeito da infusao de soluçao cardioplégica cristalóide a altas pressoes sobre a funçao endotelial de artérias epicárdicas de caes. Nao se encontraram alteraçoes a nível de receptores (curvas dose-respostas à ACH e ADP; da transduçao do sinal iniciado nos receptores/sitema de G-proteínas (fluoreto de sódio) e nos processos intracelulares da produçao de EDRF/NO (fosfolipase C e ionóforo do cálcio A23187). A funçao da musculatura lisa vascular nao foi afetada quando se analisaram as respostas relaxantes (nitroprussiato de sódio e isoproterenol) e contráteis (KCI e prostaglandina 2alfa). Estes achados permitem as seguintes consideraçoes especulativas: a) O barotrauma produzido pela infusao da cardioplegia cristalóide a altas pressoes ocorreria apenas em circulaçoes coronarianas previamente doentes? b) Uma vez que as infusoes duraram de 2 a 3 minutos, seria o barotrauma coronariano um fenômeno dependente do tempo de infusao? c) Para que ocorra o barotrauma seriam necessários níveis mais elevados de Potássio? d) Questionara existência do fenômeno do barotrauma coronariano produzido pela infusao de soluçoes cadioplégicas pelo menos nas condiçoes experimentais utilizadas. e) A metodologia empregada estuda apenas as reatividades vasculares de artérias coronarias epicárdicas. estas artérias seriam menos sensíveis aos efeitos da pressao de infusao da cardioplegia do que a microcirculaçao coronariana? f) Seria a circulaçao coronária do cao menos sensível a altas pressoes do que do homem? Estas observaçoes experimentais sugerem que a infusao de cardioplegia cristalóide, moderadamente hipocalêmica, a altas pressoes em um tempo de 2 a 3 minutos, nao interfere com a produçao de EDRF/NO pelo endotélio de coronárias epicárdicas do cao.

Effects of calcium antagonists on contractions of chorionic arteries in normal and preeclampsia placenta

This study was undertaken to observe the effects of organic or inorganic calcium antagonists and to investigate the involvement of cyclic nucleotides in regulating the vascular tone in the chorionic artery from normal or preeclamptic placenta. KCI and prostaglandin (PG) F2 alpha produced marked and constant contractions in chorionic arterial preparations of both normal and preeclamptic placentas. Nifedipine (NIF), verapamil (VER) and diltiazem (DIL) reduced the tension that had been produced by KCI and PGF2 alpha in a concentration-dependent fashion in both preparations, and the potency order of the three agents was NIF> VER > DIL. In preeclamptic arteries, however, the magnitudes of vasodilatation induced by NIF and DIL were much smaller than those in normal chorionic arteries. Mg2+ and Cd2+ also relaxed the tension induced by KCI and PGF2 alpha. In preeclamptic chorionic artery, the vasodilatation induced by Mg2+ was significantly potentiated, while that by Cd2+ was not. Removing endothelium did not alter cyclic GMP content in both preparations. In both preparations contracted by PGF2 alpha, nitroprusside markedly increased cyclic GMP content, but neither cyclic GMP nor cyclic AMP content was affected by acetylcholine, NIF, isopro-terenol, or Mg2+. The above results suggest that neither cyclic AMP nor cyclic GMP is involved in regulating the vascular tone of chorionic artery and that sensitivity of the artery in preeclampsia to the inhibitory action of calcium antagonist might be different from that in normal placenta.

Clinical experience in the use of prostin E2 [Dimoproston] vaginal tablet in obstetric department, Liaquat medical college hospital

Prostaglandins are now widely used in Obstetrics for terminating pregnancy at any stage of gestation. The efficacy and acceptability of protaglandin E and vaginal Tablet was assessed over a period of one year in 120 patients aged 1 4-42 years. The object was to achieve delivery, preferably vaginally. One hundred and ten patients delivered vaginally and 10 needed operative delivery. This preparation was well tolerated with minimum side-effects. The use of Prostin E2 vaginal Tablet is strongly recommended because of its high efficacy, acceptability and minimum patient interference

Mechanism of aspirin induced neural tube defect in chick embryo.

The effect of acetyl salicylic acid (aspirin) on neural tube development in chick embryo was studied, using the chick embryo blastoderm model. Aspirin was injected in four different doses sub-blastodermally into fresh embryonated eggs. The role of PGE1 and PGE2 alpha in the defect induced by aspirin on neural tube development in chick embryo was studied. PGE1 (5 micrograms) given after aspirin (30 micrograms) treatment was found to produce greater defect in development. All the four doses of aspirin used (i.e., 6, 30, 60 and 120 micrograms/embryo) produced significant changes (P < 0.01) in the neural tube development of chick embryo. Pre-treatment with PGE1 did not modify the defect induced by aspirin, whereas pre-treatment with PGF2 alpha prevented neural tube defects induced by aspirin. It appears that aspirin (in the doses used) affects neural tube formation by decreasing PGF2 alpha synthesis in chick embryo blastoderm.

comparison between the effect of calcium channel blocking agents and salbutamol on pregnant rabbit with reference to antagonism with oxytocin and prostaglandin F2a

The effect of Ca[2+] channels blocking agents verapamil and diltiazem either alone or in combination with oxytocin or prostaglandin F[2] alpha on pregnant rabbit's uterus near full term was studied in vivo. Verapamil [0. 25 mg/kg] produced significant decrease in the amplitude while diltiazem [0.5 mg/kg] produced significant decrease in both the amplitude and frequency of the uterine contractions with no effect on mean blood pressure. When the doses were doubled, both verapamil and diltiazem produced signficiant decrease in the amplitude and frequency of uterine contractions as well as significant decrease in the mean blood pressure. Although qualitatively similar results were obtained for salbutamol, the effects of the three drugs are different quantitatively. Their effects can be arranged in a decreasing order to be diltiazem > verapamil > salbutamol on the uterus and salbutamol > verapamil > diltiazem on systemic BP. In another set of experiments, verapamil [0.5 mg/kg] and diltiazem [1 mg/kg] were administered 5 minutes either before or after oxytocin as well as prostaglandins. Diltiazem completely abolished both oxytocin and prostaglandin F[2] alpha induced uterine activities while verapamil only partially did so. It can be concluded that Ca[2+] channel blockers can decrease the uterine activities and this effect can be used therapeutically to delay premature labour

Regulation of estrous cycle in hysterectomized albino rats by activation of follicular development.

Wistar strain albino rats were hysterectomized and the estrous cycle was compared with sham operated controls. Duration of estrous cycle in hysterectomized rats increased markedly with significant delay in the luteal phase and this was correlated to the inhibited follicular development of ovary. When these rats were treated with PGF2 alpha and PMSG and subjected to physical exercises, the estrous cycle was synchronised and the ovaries of such animals had active follicular development. Thus the deranged operation of sexual cycle in hysterectomized rats was regulated through physical exercises.

Regulation of hysterectomy induced derangements in ovarian carbohydrate metabolism in albino rats. II. Role of prostaglandin F2 alpha.

Wistar strain albino rats were subjected to bilateral hysterectomy surgically and the ovarian carbohydrate metabolism of these animals was compared with sham operated controls. The ovarian glycogen content of hysterectomized animals was elevated with inhibition of glycogenolysis, hexose mono and diphosphate pathways and oxidative metabolism. Administration of PGF2 alpha to hysterectomized animals led to activation of ovarian glycogenolysis and other pathways of carbohydrate metabolism of hysterectomized animals was restored towards normal level after PGF2 alpha substitution.