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.

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.

Effects of recombinant bovine somatotropin on pregnancy per artificial insemination, corpus luteum cellular composition and endometrial gland morphometry in beef cattle.

The aim of this research was to evaluate the effect of recombinant bovine somatotropin (bST) on pregnancy per artificial insemination (P/AI), cellular composition of the corpus luteum (CL) and endometrial gland morphometry. In Experiment 1, Nelore cows (n = 587) received a fixed-time artificial insemination (FTAI) protocol and, at insemination, received 0, 250 or 500 mg of bST subcutaneously (SC). In Experiment 2, Nelore cows (n = 243) received 0 or 500 mg of bST, SC, on D7 (D0 = day of FTAI). Blood samples were collected on D7 and D16 to measure progesterone (P4) concentrations. In Experiments 1 and 2, pregnancy diagnosis was performed 30 days after FTAI. In Experiment 3, Nelore heifers (n = 20) received a FTAI protocol, but were not inseminated, and on D0 (ovulation day), they received 0 (bST 0; n = 9) or 500 mg of bST (bST 500; n = 11), SC. The heifers were slaughtered on D15 (D0 = ovulation day), at which time the CL was evaluated for diameter, weight, a percentage of large (LLC) and small (SLC) luteal cells, and the concentration of progesterone in plasma measured. The number, perimeter and area of superficial and deep endometrial glands were evaluated. There was no difference in P/AI when bST was applied on D0 and D7. In Experiment 1, P/AI did not differ among treatments, with 59.28% (115/194), 58.38% (115/197) and 65.82% (129/196) for the bST 0, 250 and 500 treatments, respectively. In Experiment 2, P/AI did not differ between treatments, with 57.3% (71/124) and 60.5% (62/119) for the bST 0 and 500 treatments, respectively. Plasma progesterone concentrations on D16 was greater in the bST 500 (11.63 ±â€¯0.84 ng/mL) than bST 0 (9.83 ±â€¯0.88 ng/mL). In Experiment 3, there was no difference in ovarian diameter and weight, CL diameter, percentage of SLC, P4 concentrations and endometrial gland morphology. Heifers in the bST 500 treatment had heavier CL (3.11 ±â€¯0.32 vs. 2.25 ±â€¯0.20 g); however, the bST 0 treatment heifers had a greater percentage of LLC than did the bST 500 treatment (13.72 ±â€¯1.16% vs. 8.60 ±â€¯1.52). It was concluded that the doses of bST used in this study do not increase P/AI; however, they do cause changes in P4 concentration and the cellular composition of the CL.

Influence of male on estrus behavior, estradiol and progesterone release and puberty onset on prepubertal Saanen goats / Influência do reprodutor sobre o comportamento de estro, liberação de estrógeno, progesterona e início da puberdade em cabras Saanen pré-puberes

The present study investigated the male effect on the estrus behaviors, estradiol and progesterone release in prepubertal Saanen goats. Twenty-nine female Saanen goats at 135 ± 10 days old with body weight of 22.8 ± 3.3 Kg were randomly assigned to three treatments: exposure to sexually active male (male treatment), exposure to androgenized females (androgenized female treatment), and prepubertal goats isolated from active male and androgenized females (control treatment). Sexual behaviors associated with estrus were recorded daily, and blood samples were taken weekly to determine estradiol and progesterone concentrations over 24 weeks. The experimental goats subjected to male or androgenized female had significantly higher frequency of estrus (mount acceptance) (P ≤ 0,02), progesterone (P ≤ 0,01), and estradiol (P ≤ 0,01) release than the control goats. Furthermore, goats exposed to a male showed estrus behavior two weeks earlier and maintained this estrus behavior for three weeks more than goats of both female and control treatments. Estrus was observed in 70 % of goats in male and female treatments during the breeding season versus 44 % of the control goats. Finally, significantly more goats subjected to male treatment (60 % of goats) showed progesterone concentrations higher than 1 ng mL-1 (which indicates the presence of a functional corpus luteum) compared to the female and control treatment (40 and 22 % of goats, respectively). These results shows that male treatment significantly increased the number of females showing estrus behavior, estradiol and progesterone release, and the number of animals with a functional corpus luteum, anticipating puberty for experimental goats, suggesting that the male effect could be used to anticipate the onset of puberty in goats.(AU)

O presente estudo investigou o efeito do macho sobre o comportamento do estro, liberação de estradiol e progesterona em cabritas Sannen pré-púberes. Vinte e nove cabritas com 135 ± 10 dias de idade e peso corporal de 22,8 ± 3,3 kg foram submetidas à três tratamentos: macho; fêmeas androgenizadas; controle (mantidas isoladas do efeito macho ou de fêmeas androgenizadas) . Os comportamentos sexuais foram registrados diariamente e as amostras de sangue foram colhidas semanalmente ao longo de 24 semanas. Os tratamentos macho e fêmea androgenizada aumentaram significativamente a ocorrência comportamental do estro (P ≤ 0,02), a concentração de progesterona (P ≤ 0,01) e estradiol (P ≤ 0,01) em comparação ao tratamento controle. As cabritas expostas ao efeito macho anteciparam o comportamento de estro em duas semanas, e o mantiveram por mais três semanas quando comparado às cabritas dos tratamentos fêmea androgenizada e controle. Apenas 44% das cabritas controle foram observadas em estro, sendo que 70% das cabritas submetidas aos tratamentos macho e fêmea androgenizada foram observadas em estro. Além disso, 60% das cabritas expostas ao efeito macho, 40 % das cabritas expostas ao efeito fêmea androgenizada e 22% das cabritas controle apresentaram concentrações de progesterona superiores a 1 ng mL-1, o que indica a presença de corpo lúteo funcional. De fato, o efeito macho aumentou significativamente o número de fêmeas em estro, a concentração de estradiol e progesterona, o número de fêmeas com corpo lúteo funcional, sugerindo que o efeito macho pode ser usado para antecipar o início da puberdade em cabritas Saanen.(AU)

Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary.

BACKGROUND: Anti-Müllerian hormone (AMH) is expressed by granulosa cells of developing follicles and plays an inhibiting role in the cyclic process of follicular recruitment by determining follicle-stimulating hormone threshold levels. Knowledge of AMH expression in the porcine ovary is important to understand the reproductive efficiency in female pigs. RESEARCH AIM: In the present study we investigated the expression of AMH during follicular development in prepubertal and adult female pigs by immunohistochemistry, laser capture micro-dissection and RT-qPCR. RESULTS AND CONCLUSION: Although in many aspects the immunohistochemical localization of AMH in the porcine ovary does not differ from other species, there are also some striking differences. As in most species, AMH appears for the first time during porcine follicular development in the fusiform granulosa cells of recruited primordial follicles and continues to be present in granulosa cells up to the antral stage. By the time follicles reach the pre-ovulatory stage, AMH staining intensity increases significantly, and both protein and gene expression is not restricted to granulosa cells; theca cells now also express AMH. AMH continues to be expressed after ovulation in the luteal cells of the corpus luteum, a phenomenon unique to the porcine ovary. The physiological function of AMH in the corpus luteum is at present not clear. One can speculate that it may contribute to the regulation of the cyclic recruitment of small antral follicles. By avoiding premature exhaustion of the ovarian follicular reserve, AMH may contribute to optimization of reproductive performance in female pigs.

Equine Chorionic Gonadotropin Modulates the Expression of Genes Related to the Structure and Function of the Bovine Corpus Luteum.

We hypothesized that stimulatory and superovulatory treatments, using equine chorionic gonadotropin (eCG), modulate the expression of genes related to insulin, cellular modelling and angiogenesis signaling pathways in the bovine corpus luteum (CL). Therefore, we investigated: 1-the effect of these treatments on circulating insulin and somatomedin C concentrations and on gene and protein expression of INSR, IGF1 and IGFR1, as well as other insulin signaling molecules; 2-the effects of eCG on gene and protein expression of INSR, IGF1, GLUT4 and NFKB1A in bovine luteal cells; and 3-the effect of stimulatory and superovulatory treatments on gene and protein expression of ANG, ANGPT1, NOS2, ADM, PRSS2, MMP9 and PLAU. Serum insulin did not differ among groups (P = 0.96). However, serum somatomedin C levels were higher in both stimulated and superovulated groups compared to the control (P = 0.01). In stimulated cows, lower expression of INSR mRNA and higher expression of NFKB1A mRNA and IGF1 protein were observed. In superovulated cows, lower INSR mRNA expression, but higher INSR protein expression and higher IGF1, IGFR1 and NFKB1A gene and protein expression were observed. Expression of angiogenesis and cellular modelling pathway-related factors were as follows: ANGPT1 and PLAU protein expression were higher and MMP9 gene and protein expression were lower in stimulated animals. In superovulated cows, ANGPT1 mRNA expression was higher and ANG mRNA expression was lower. PRSS2 gene and protein expression were lower in both stimulated and superovulated animals related to the control. In vitro, eCG stimulated luteal cells P4 production as well as INSR and GLUT4 protein expression. In summary, our results suggest that superovulatory treatment induced ovarian proliferative changes accompanied by increased expression of genes providing the CL more energy substrate, whereas stimulatory treatment increased lipogenic activity, angiogenesis and plasticity of the extracellular matrix (ECM).

A link between Notch and progesterone maintains the functionality of the rat corpus luteum.

In this study, we investigated the interaction between the Notch pathway and progesterone to maintain the functionality of the corpus luteum (CL). When Notch signaling is activated, the γ-secretase complex releases the active intracellular domains (NICD) of their receptors, which exert survival effects. We designed studies to analyze whether the in vitro inhibition of Notch affects progesterone production, steroidogenic regulators, apoptotic parameters, and signaling transduction pathways in the cultures of CL isolated from pregnant and superovulated rats. We detected a decrease in progesterone production when corpora lutea (CL) were incubated with N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor. This effect could be in part due to the decrease detected in the CL protein levels of P450scc because STAR and 3ß-hydroxysteroid dehydrogenase were not affected by Notch inhibition. Besides, the addition of aminoglutethimide to the CL culture medium decreased NICD of NOTCH1. We observed an increase in the expression of active CASPASE3 (CASP3) after inhibition by Notch, which was reversed by the presence of progesterone. The BAX:BCLXL ratio was increased in CL treated with DAPT and the presence of progesterone reversed this effect. In addition, phosphorylation of AKT was inhibited in CL treated with DAPT, but had no effect on ERK activation. To demonstrate that the action of DAPT is specifically related with the inhibition of Notch, CLs were incubated with DLL4 antibody and a decrease in progesterone production was detected. These results suggest the existence of a novel link between progesterone and the Notch signaling pathway to maintain the functionality of the CL.

Role of ghrelin in fertilization, early embryo development, and implantation periods.

In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4-71.4% and antagonist, 75.0-62.5% vs ghrelin 2, 46.2-15.4% and control, 10-0.0%; n=7-13 females/group; P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%; n=82-102 embryos/treatment; P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in 'adequate' concentrations) has a physiological role in early gestational events.

Oogenesis and ovarian histology in two populations of the viviparous lizard Sceloporus grammicus (Squamata: Phrynosomatidae) from the central Mexican Plateau.

The annual histological changes in ovarian morphology (oogenesis, follicular atresia, and corpus luteum) are described for the Mexican lizard Sceloporus grammicus, in two populations that inhabit contrasting environments (vegetation categories, climate, precipitation, and temperature) from Hidalgo State, Mexico. Two germinal beds were situated on the dorsal surface of each ovary of this species. In both the populations, oogenesis involves two major processes: previtellogenesis and vitellogenesis. The histological changes during previtellogenesis are similar to those for other reptilian sauropsids, whereas vitellogenesis differs and the features of this last process are described for the first time. In early previtellogenesis, primary oocytes have fibrillar chromosomes and the ooplasm stains slightly. The primordial follicles are surrounded by a granulosa composed of cuboidal follicular cells. During late previtellogenesis, the oocyte had an eccentric nucleus with lamp-brush chromosomes and multiple nucleoli. The granulosa becomes multilayered and polymorphic, containing three cell types: small, intermediate, and pyriform. The zona pellucida was homogeneous and clearly observed. In early vitellogenesis, the oocyte showed several small acidophilic granules distributed in the center and the periphery of the oocyte. As vitellogenesis progresses, the yolk platelets move toward the central area of the oocyte and they fuse to form acidophilic and homogeneous yolk. Lipid droplets were distributed irregularly in the ooplasm of the oocyte. In Zacualtipán, the results revealed a strong seasonal reproductive activity. Females had vitellogenic follicles from July to September, and pregnant females were founded from September to March. In Tizayuca, the results showed an unusual pattern of reproductive activity. Females with vitellogenic follicles and pregnant females were found throughout the year, indicating continuous reproduction. We suggest that the observed differences in reproductive activity from these populations indicate adaptative fine tuning in response to local environmental conditions. These results contribute to the knowledge of variation in vitellogenesis and reproductive strategies of this species and among spiny lizards overall.

Estrogen and oxytocin receptors in the canine corpus luteum during pregnancy and parturition / Receptores de estrógeno e ocitocina no corpo lúteo durante a gestação e parto em cadelas

The expression of genes encoding the receptors for estrogen (ERαmRNA) and oxytocin (OTRmRNA) was studied in the corpus luteum during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERαmRNA and OTRmRNA in the corpus luteum of bitches during Early (up to 20 days of gestation), Mid (20 to 40 days) and Late Pregnancy (40 to 60 days), and Parturition (first stage of labor). The corpus luteum expressed mRNA for OTR, however ERα mRNA was not detected. There was a reduction of OTR mRNA expression in the corpus luteum from gestational Day 20 onward, which suggests an important role of OTR mRNA in the mechanism of pregnancy recognition in dogs. We concluded that the expression of OTR mRNA in canine corpus luteum vary over time, which support the idea that the sensitivity and response to hormone therapy can vary along the course of pregnancy and labor. Moreover, the canine CL lacks ERα mRNA expression during pregnancy.(AU)

A expressão dos genes que codificam os receptores de estrógeno (REα RNAm) e ocitocina (ROT RNAm) foi estudado no corpo lúteo de cadelas durante a gestação e parto. A técnica de PCR em tempo real foi realizada para quantificar a expressão do REα RNAm e ROT RNAm no corpo lúteo de cadelas durante o início (até 20 dias de gestação), meio (20 a 40 dias) e final da gestação (40 a 60 dias), e durante o parto (pródomos do parto). O corpo lúteo apresentou expressão do RNAm para o ROT, entretanto o RNAm para o REα não foi detectado. Houve redução na expressão do ROT RNAm no corpo lúteo a partir de 20 dias da gestação, indicando papel no mecanismo de reconhecimento gestacional em cadelas. Em conclusão, a expressão do ROT RNAm no corpo lúteo de cadelas apresentou variação ao longo do tempo de gestação, sugerindo que a resposta e sensibilidade à terapia hormonal pode variar conforme o momento da gestação e parto. Ademais, o corpo lúteo canino não expressa REα RNAm durante a gestação.(AU)

Estrogen and oxytocin receptors in the canine corpus luteum during pregnancy and parturition / Receptores de estrógeno e ocitocina no corpo lúteo durante a gestação e parto em cadelas

The expression of genes encoding the receptors for estrogen (ERαmRNA) and oxytocin (OTRmRNA) was studied in the corpus luteum during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERαmRNA and OTRmRNA in the corpus luteum of bitches during Early (up to 20 days of gestation), Mid (20 to 40 days) and Late Pregnancy (40 to 60 days), and Parturition (first stage of labor). The corpus luteum expressed mRNA for OTR, however ERα mRNA was not detected. There was a reduction of OTR mRNA expression in the corpus luteum from gestational Day 20 onward, which suggests an important role of OTR mRNA in the mechanism of pregnancy recognition in dogs. We concluded that the expression of OTR mRNA in canine corpus luteum vary over time, which support the idea that the sensitivity and response to hormone therapy can vary along the course of pregnancy and labor. Moreover, the canine CL lacks ERα mRNA expression during pregnancy.

A expressão dos genes que codificam os receptores de estrógeno (REα RNAm) e ocitocina (ROT RNAm) foi estudado no corpo lúteo de cadelas durante a gestação e parto. A técnica de PCR em tempo real foi realizada para quantificar a expressão do REα RNAm e ROT RNAm no corpo lúteo de cadelas durante o início (até 20 dias de gestação), meio (20 a 40 dias) e final da gestação (40 a 60 dias), e durante o parto (pródomos do parto). O corpo lúteo apresentou expressão do RNAm para o ROT, entretanto o RNAm para o REα não foi detectado. Houve redução na expressão do ROT RNAm no corpo lúteo a partir de 20 dias da gestação, indicando papel no mecanismo de reconhecimento gestacional em cadelas. Em conclusão, a expressão do ROT RNAm no corpo lúteo de cadelas apresentou variação ao longo do tempo de gestação, sugerindo que a resposta e sensibilidade à terapia hormonal pode variar conforme o momento da gestação e parto. Ademais, o corpo lúteo canino não expressa REα RNAm durante a gestação.

Análise diferencial da expressão gênica e proteica no corpo lúteo de bovinos submetidos a tratamentos com eCG / Differential analysis of the gene and protein expression in bovine corpus luteum under eCG treatments

A gonadotrofina coriônica equina (eCG) tem sido utilizada em programas de sincronização para inseminação artificial em tempo fixo e normalmente promove o aumento do volume do corpo lúteo e a da produção de progesterona. Além disso, esta mesma gonadotrofina pode ser utilizada para superovulação. Desse modo, hipóteses relativas aos mecanismos pelos quais gonadotrofinas exógenas alteram as funções celulares nos corpos lúteos resultantes foram formuladas. Para testar tais hipóteses, 18 vacas (Bos indicus) foram divididas em grupos: controle (n=5), estimulado (n=6) e superovulado (n=7) e a ovulação das mesmas foi sincronizada usando um protocolo já estabelecido com dispositivo de progesterona. Os animais estimulados receberam 400 UI de eCG no dia de remoção do dispositivo de progesterona e os animais superovulados 4 dias antes. No dia 7 após injeção de GnRh, os animais foram abatidos para a coleta de CLL e sangue. Análises de peso e volume de CL, concentração de progesterona (P4), bem como da expressão gênica e proteica de fatores angiogênicos e de proteínas esteroidogênicas foram realizadas. Além disso, o transcriptoma foi analisado por microarranjo. Foi observado que o volume do CL foi maior nos animais do grupo estimulado (1177,37 ± 167,07 mm3) e ainda maior nos do superovulado (1495,18 ± 137,01 mm3) quando comparados ao grupo controle (830,33 ± 234,99 mm3; p = 0,03). A concentração média de progesterona por CL nos animais do grupo estimulado foi maior que nos animais do grupo controle (5,95 ± 0,17 vs 3,69 ± 0,72 ng/ml; p = 0,03) e que nos superovulados (4,11 ± 0.73; p = 0,01). Além disso, os tratamentos com eCG aumentaram a expressão do FGFR2 e também da STAR nos animais estimulados e superovulados (p < 0,05). Quanto aos resultados do microarranjo, no total 242 transcritos foram aumentados e 111 foram diminuídos nos animais estimulados e 111 foram aumentados e 113 diminuídos nos animais superovulados em relação aos animais controle (1,5 vezes, p 0.05).

Equine chorionic gonadotropin (eCG) has been widely used in synchronization protocol to artificial insemination program and usually promote corpus luteum (CL) volume increases and stimulates progesterone production. Furthermore the same gonadotropin can be used to superovulation protocols. Thus, hypotheses concerning the mechanisms by which exogenous gonadotropins alter cellular functions in resulting corpora lutea were formulated. To test that hypothesis, 18 (Bos indicus) cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronized using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG of device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea (CLL) and blood samples were collected seven days after GnRH administration. Analyses of CL weight and volume, progesterone (P4) concentration, as well as the gene and protein expression of angiogenic and steroidogenic proteins were performed. Furthermore, the transcriptome was evaluated by microarray. The CL volume was higher in superovulated (1495.18 ± 137.01) than in stimulated (1177.37 ± 167.07) cows and higher in stimulated than in the control (830.33 ± 234.99) cows, and the P4 concentration per CL was higher in stimulated (5.95 ± 0.17 ng/ml) animals than in the control (3.69 ± 0.72 ng/ml) and superovulated (4.11 ± 0.73 ng/ml; P = 0.01) animals. Overall, 242 transcripts were up-regulated and 111 transcripts were downregulated in stimulated cows (P 0.05) and 111 were up-regulated and 113 down-regulated in superovulated cows in relation to the control (1.5 fold, P 0.05).

Allopregnanolone alters the luteinizing hormone, prolactin, and progesterone serum levels interfering with the regression and apoptosis in rat corpus luteum.

Steroids synthesized in the central nervous system are termed "neurosteroids". They are synthesized and metabolized in several brain areas. The objective of this work was to determine if 1 intracerebroventricular allopregnanolone injection in rats can interfere in luteal regression in a close relationship with modifications in LH, progesterone, and prolactin serum concentrations. Allopregnanolone was injected during proestrus morning and the animals were sacrificed on oestrous morning. Ovulation test and histological analysis were performed in the oestrus morning with light and electron microscopy. Serum prolactin, LH, and progesterone levels were measured by radioimmunoassay. The allopregnanolone injection significantly decreased luteinizing hormone serum level and the number of oocytes on oestrus. Progesterone and prolactin serum levels were increased after this injection. The inhibition of apoptotic figures due to allopregnanolone administration was detected in the already formed corpora lutea belonging to the previous ovary cycle and it was significantly lower than in vehicle group (control). When the GABA(A) antagonist (bicuculline) was administered alone or previously to allopregnanolone, no effect on the ovulation rate was observed. No changes in the apoptotic cell numbers were observed with respect to those of vehicle group. These results show that the effect of centrally injected allopreganolone over reproductive function could be due to a centrally originated LH mediated effect over ovarian function that affects luteal regression, through the inhibition of apoptosis and stimulation of progesterone and prolactin release.

Efeito da aplicação de eCG ou hCG 7 dias após a IATF no desenvolvimento das estruturas ovarianas e nas taxas de prenhez de vacas de corte / Effect of administration of eCG or hCG 7 days after FTAI on the development of ovarian structures and pregnancy rates in beef cows

Background: The early embryo development is affected by the progesterone concentration, especially on the first weeks after conception. It's well known that the size and weight of corpus luteum (CL) is positive correlated with higher progesterone production. The use of treatments that increase luteal function in different periods after fixed timed insemination (FTAI) has been tested recently, aiming better embryo survival rates and pregnancy establishment. The objective of this experiment was evaluate the effect of treatment with hCG or eCG 7 days after FTAI on the development of the CL, follicle growth and pregnancy rate in beef cows. Materials, Methods & Results: Two hundred and nineteen Brangus cows were synchronized to FTAI using intravaginal implants containing 1g of progesterone for 8 days and injecting 2 mg of estradiol benzoate (EB), on the first day of treatment. All cows received 150 mcg of D-Cloprostenol, i.m., on Day 8 and 24 h after, 1 mg of EB, i.m. The timed AI were done 52-56 h after the implant removal. Seven days after the AI the cows were randomly assigned in 3 different groups according the treatment. hCG (n = 40) receiving 1500 IU of hCG, i.m.; eCG (n = 41) injection of 400 IU of eCG, i.m., and Control (n = 138). The ultrasonographic examinations were done on days 0, 7 and 12 to determine the presence and diameter of follicles, area of CL on the subgroups hCG (n = 26), eCG (n = 26) and Control (n = 14). The pregnancy diagnosis was done 30 days after FTAI and another exam was done 60 days after FTAI only on the cows that were pregnant on day 30 to verify the embryonic loss. The statistical analyses were performed using the SPSS for Windows. Pregnancy rates were compared by chi-square test, other parameters by ANOVA, the means were compared by Tukey test. The pregnancy rates of FTAI were 60.98% (25/41), 45% (18/40) and 40.58% (56/138) to eCG, hCG and Control, respectively, without statistic difference (P = 0.07). Even using 8 differentsires in the inseminations, the pregnancy rates were similar (P = 0.76). The size of the ovulatory follicle on the day of the FTAI didn't differ among the groups (P = 0.12). The ovulatory rate was 82%. The mean±SEM follicle diameter found on D7 didn't differ (P = 0.21), but the evaluation on D12, the diameters were 8.6 ± 4.73 (eCG), 2 ± 6.85 (hCG); 3.74 ± 4.7 mm (Control) and differed among the groups (P < 0.01). The CL area (cm²) on D7 was similar for the groups (P = 0.21), on D12 the areas were 3.05 ± 1.22; 3.97 ± 1.82; 2.65 ± 1.22 to eCG, hCG and Control group respectively (P = 0.014). It was observed the presence of a CL with area higher than 2 cm² in 80.8% of the cows of the groups eCG and hCG, meanwhile the Control group had only 54.2%. The control group had the lowest CL growth rate, 0.23 ± 0.52 cm²/day, the eCG treated had intermediary growth, 1.01 ± 1.17 cm²/day and the ones that received hCG had the highest daily growth (1.32 ± 1.63 cm²/day). Discussion: Although 20 percentage points above in the cows treated with eCG in comparison to the Control group, the pregnancy rates were statistically similar. Results indicate that treating cows with hCG 7 days after the FTAI promotes improved size of CL, induced ovulation of follicles between days 7 and 12 after FTAI causing the development of accessory CL, and the use of eCG stimulates growth of CL and follicles.

[Relevance of apoptosis in the female reproductive system]. / Relevancia de la apoptosis en la reproducción femenina.

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.

Relevancia de la apoptosis en la reproducción femenina / Relevance of apoptosis in the female reproductive system

La apoptosis es un proceso genéticamente controlado mediante el cual las células inducen su propia muerte. Mensualmente y en forma cíclica, el ovario, el endometrio y la glándula mamaria atraviesan por ciclos de proliferación celular y apoptosis respondiendo a los cambios en la secreción hormonal. Durante el desarrollo embrionario, la apoptosis está implicada en procesos relacionados con la escultura de los diferentes órganos, a través de la eliminación de estructuras innecesarias y con el control de las células defectuosas. Asimismo, la apoptosis juega un papel fundamental en la función ovárica. La reserva folicular se establece durante la vida fetal y luego se va eliminando gradualmente. La apoptosis está involucrada tanto en la muerte celular durante el proceso de reclutamiento del folículo dominante, como en la luteólisis. Durante la pubertad la apoptosis contribuye a la formación del espacio luminal de los ductos terminales de la mama. A su vez, el proceso de involución mamaria luego de la lactancia se caracteriza por una apoptosis masiva de las células epiteliales secretoras. Así como la apoptosis está involucrada en los cambios fisiológicos que ocurren a nivel endometrial, también se ha asociado a la muerte celular programada con procesos patológicos, especialmente en aquellos caracterizados por el incremento en el crecimiento celular como es el caso de la endometriosis. El delicado balance entre la apoptosis y la proliferación celular es fundamental ya que permite que los tejidos puedan responder en forma cíclica a los cambios hormonales fisiológicos y prevenir procesos de transformación neoplásica.

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat. A freeze-fracture study.

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat: a freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat: a freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe en face the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.(AU)

Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira).

This study aimed to establish a protocol for synchronization of estrus in brown brocket deer (Mazama gouazoubira). Two groups of hinds (n=3) were submitted to two different protocols: Treatment 1 received an intravaginal progesterone (CIDR) device for 8 days, followed by 265microg injection of cloprostenol at the time of removal; and Treatment 2 received two injections of 265microg of cloprostenol 11 days apart. After 30 days, each group of three hinds received the other treatment. Treatment efficacy was evaluated by reproductive behavior, fecal progestin and estrogen concentration and the observation of CL by laparoscopy 6 days after the end of estrus. All the hinds (100%) had estrous behavior upon the completion of treatment, but a significant difference occurred between the time of onset, 70.5+/-5.0h for Treatment 1 and 52.3+/-5.6h for Treatment 2. The mean estrus duration time (34.7+/-4.50 and 37.0+/-8.11h), ovulation rates (5/6 and 4/6), mean CL size (4.85+/-0.74 and 3.21+/-0.19mm) and mean fecal progestin concentration at 6 days after the end of estrus (865.53+/-76.59 and 1073.35+/-106.82ng/g feces) were not significantly different between treatments. There was no difference in fecal estrogen concentrations throughout the treatment and the greatest values of the estrogen:progestin ratio coincided with estrous behavior. Although fertility was not evaluated directly, both treatments were effective in synchronizing estrus in the species M. gouazoubira, with the formation of functional corpora lutea.