Prolonged use of letrozole causes morphological changes on gonads in Galea spixii

Abstract Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of long-term use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix's yellow-toothed cavy; Galea spixii). Forty-eight rodents (24 males and 24 females) were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.

Prolonged use of letrozole causes morphological changes on gonads in Galea spixii

Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of longterm use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix's yellow-toothed cavy; Galea spixii). Forty-eight rodents (24 males and 24 females) were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.(AU)

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Effect of recombinant bovine somatotropin (rbST) treatment on follicular population and development in non-lactating dairy cows

The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.(AU)

Granulosa cell tumor associated with occult hyperadrenocorticism in a yorkshire terrier bitch

Background: Hyperadrenocorticism (HAC), is considered a set of symptoms due to excessive exposure to cortisol. Naturally occurring HAC is most often related to pituitary tumors (pituitary-dependent HAC - PDH). Occult HAC, is referred as a clinical picture highly consistent with HAC; however, routine screening tests are negative. In addition, one or more steroids are elevated following administration of adrenocorticotrophic hormone (ACTH). Ovarian granulosa cell tumors, can produce steroids leading to paraneoplastic syndromes. The objective of this study was to report an unpublished case of ovarian granulosa cell tumor associated with occult hyperadrenocorticism in a Yorkshire Terrier.Case: A 13-year-old intact female dog, Yorkshire Terrier, was brought for consultation with slight weight loss, polyuria, polydipsia, irregular estrous cycles, increased abdominal volume and thin coat. On physical examination the animal was gasping and presented severe periodontal disease, bulging abdomen, alopecia and skin hyperpigmentation. Complete blood count presented no changes; however, serum biochemistry evaluation highlighted hyperalbuminemia, hypertriglyceridemia, alkaline phosphatase increased activity, and urinary specific gravity and creatinine below reference values. On abdominal ultrasonography left adrenal gland measured 2.08 cm x 1.08 cm and the right adrenal gland measured 2.11 cm x 0.84 cm, indicating bilateral adrenomegaly compatible with PDH. In the hypogastric abdomen, a large heterogeneous hypoechogenic mass was also observed, with areas of cystic cavities, measuring 5.80 cm x 7.30 cm. A low dose dexamethasone suppression test (LDDST) was performed, due to PDH suspicion. The test did not confirm HAC, suspecting, then, to be a case of occult/atypical HAC. Due to the strong clinical suspicion, and owner financial problems for further investigated occult HAC, trilostane treatment was initiated.[...](AU)

Granulosa cell tumor associated with occult hyperadrenocorticism in a yorkshire terrier bitch

Background: Hyperadrenocorticism (HAC), is considered a set of symptoms due to excessive exposure to cortisol. Naturally occurring HAC is most often related to pituitary tumors (pituitary-dependent HAC - PDH). Occult HAC, is referred as a clinical picture highly consistent with HAC; however, routine screening tests are negative. In addition, one or more steroids are elevated following administration of adrenocorticotrophic hormone (ACTH). Ovarian granulosa cell tumors, can produce steroids leading to paraneoplastic syndromes. The objective of this study was to report an unpublished case of ovarian granulosa cell tumor associated with occult hyperadrenocorticism in a Yorkshire Terrier.Case: A 13-year-old intact female dog, Yorkshire Terrier, was brought for consultation with slight weight loss, polyuria, polydipsia, irregular estrous cycles, increased abdominal volume and thin coat. On physical examination the animal was gasping and presented severe periodontal disease, bulging abdomen, alopecia and skin hyperpigmentation. Complete blood count presented no changes; however, serum biochemistry evaluation highlighted hyperalbuminemia, hypertriglyceridemia, alkaline phosphatase increased activity, and urinary specific gravity and creatinine below reference values. On abdominal ultrasonography left adrenal gland measured 2.08 cm x 1.08 cm and the right adrenal gland measured 2.11 cm x 0.84 cm, indicating bilateral adrenomegaly compatible with PDH. In the hypogastric abdomen, a large heterogeneous hypoechogenic mass was also observed, with areas of cystic cavities, measuring 5.80 cm x 7.30 cm. A low dose dexamethasone suppression test (LDDST) was performed, due to PDH suspicion. The test did not confirm HAC, suspecting, then, to be a case of occult/atypical HAC. Due to the strong clinical suspicion, and owner financial problems for further investigated occult HAC, trilostane treatment was initiated.[...]

Impacto da Osteocalcina na Esteroidogenese Ovariana

A osteocalcina é conhecida como uma proteína óssea contendo ácido gama-carboxi glutâmico (BGLAP), é um pequeno (49 aminoácidos) hormônio proteico não-colágeno secretado por osteoblastos, geralmente relatado como um marcador de formação óssea. Mais recentemente, outros papéis da osteocalcina na secreção e sensibilidade à insulina foram sugeridos, embora seu impacto na secreção de esteróides ovarianos permaneça obscuro. Portanto, o presente estudo tem como objetivo investigar a presença do receptor de osteocalcina (GPCR6A) nas células da granulosa e da teca do ovário bovino e o possível efeito da osteocalcina sobre enzimas esteroidogênicas. Nossos resultados indicam, pela primeira vez, a presença do receptor osteocalcina GPRC6A nas células e testes da teca e sua ausência ou muito baixa expressão nas células da granulosa. Após o tratamento com osteocalcina não carboxilada (100ng / ml), um tamanho de efeito significativo nas principais enzimas esteroidogênicas (CYP17A1, CYP11A1) e proteína (STAR) foi observado na primeira hora (mas não 24 horas) apenas na presença de co-tratamento com hormônio luteinizante (LH). Doses mais baixas de osteocalcina não carboxilada (1 ng / ml) não foram eficazes para influenciar essas enzimas. Embora limitados, nossos resultados demonstram que parece haver alguma ação da osteocalcina na primeira hora após o tratamento, aumento a expressão de mRNA em enzimas esteroidogenicas chaves como a STAR, CYP11A1 e CYP17A1 de forma sinérgica com o uso de LH (100 ng/ml).

Osteocalcin, known as bone gamma-carboxy glutamic acid-containing protein (BGLAP), is a small (49-amino-acid) noncollagenous protein hormone secreted by osteoblasts usually reported as a marker of bone formation. More recently, other roles for osteocalcin in insulin secretion and sensibility have been suggested, although its impact on the ovarian steroid secretion remains obscure. Therefore, the present study aims to investigate the presence of osteocalcin receptor (GPCR6A) in the granulosa and theca cells of the bovine ovary and the possible effect of osteocalcin on steroidogenic enzymes. Our results indicate, for the first time, the presence of the osteocalcin receptor GPRC6A in theca cells and testis and its absence or very low expression in the granulosa cells. After treatment with non-carboxylated osteocalcin (100ng/ml), a significant effect size on the key steroidogenic enzymes (CYP17A1, CYP11A1) and protein (STAR) was observed in the first hour (but not 24-hour) only in the presence of cotreatment with luteinizing hormone (LH). Lower doses of non-carboxylated osteocalcin (1ng/ml) were not effective to influence these enzymes. Although limited, our results demonstrate that there appears to be some action of osteocalcin in the first hour after treatment, increasing mRNA expression in key steroidogenic enzymes such as STAR, CYP11A1, and CYP17A1 synergistically with the use of LH (100 ng/ml)

The role of androgens in mammals folliculogenesis / Papel dos andrógenos na foliculogênese em mamíferos

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized. Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects [...](AU)

The role of androgens in mammals folliculogenesis / Papel dos andrógenos na foliculogênese em mamíferos

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized. Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects [...]

Influência do manejo na fisiologia reprodutiva do macho bovino / Influence of management on the reproductive physiology of male bovine / Influencia del manejo en la fisiología reproductiva del bovino macho

O conhecimento da fisiologia do desenvolvimento testicular e ponderal, a precocidade sexual, a capacidade de produção e qualidade espermática, além dos fatores que potencialmente interferem nestes processos, são importantes para predizer a capacidade reprodutiva dos touros. Entre esses fatores, existem alguns que podem influenciar negativamente na fisiologia reprodutiva do touro, e, dessa forma, reduzir a fertilidade desses animais e causar esterilidade, tais como os fatores diretamente relacionados ao manejo e a nutrição. Dessa forma, torna-se de fundamental importância o conhecimento da fisiologia do desenvolvimento testicular e ponderal, e de como alguns fatores, como o manejo e o clima, podem interferir nestes processos. Portanto, o objetivo desta revisão é demonstrar como alguns fatores do ambiente, tais como o manejo e o clima, podem influenciar nessas características, com vistas à produção animal.(AU)

Knowledge of the physiology and weight of the testicular development, sexual precocity, ability and quality of sperm production, and the factors that potentially interfere with these processes are important for predicting the reproductive capacity of bulls. Among these factors, there are some that can negatively influence their reproductive physiology, and thus reduce their fertility, causing sterility, such as factors directly related to the management and nutrition. Thus, it is of fundamental importance to study the physiology of testicular development and weight, and how certain factors, such as management and climate, can interfere with these processes. Therefore, the aim of this review is to show how some environmental factors, such as management and climate, can influence these characteristics, aiming to animal production.(AU)

El conocimiento de la fisiología del desarrollo testicular y ponderal, la precocidad sexual, la capacidad de producción y la calidad del esperma, además de los factores que potencialmente interfieren en estos procesos, son importantes para predecir la capacidad reproductiva de los toros. Entre estos factores, hay algunos que pueden influir negativamente en la fisiología reproductiva del toro, y por lo tanto reducir la fertilidad de esos animales y causar esterilidad, tales como los factores directamente relacionados al manejo y la nutrición. Por lo tanto, es de fundamental importancia el conocimiento de la fisiología del desarrollo testicular y ponderal, y cómo ciertos factores, como el manejo y el clima, pueden interferir en estos procesos. Así, el objetivo de esta revisión es demostrar cómo algunos factores ambientales, tales como manejo y el clima, pueden influenciar en esas características, con miras a la producción animal.(AU)

Influência do manejo na fisiologia reprodutiva do macho bovino / Influence of management on the reproductive physiology of male bovine / Influencia del manejo en la fisiología reproductiva del bovino macho

O conhecimento da fisiologia do desenvolvimento testicular e ponderal, a precocidade sexual, a capacidade de produção e qualidade espermática, além dos fatores que potencialmente interferem nestes processos, são importantes para predizer a capacidade reprodutiva dos touros. Entre esses fatores, existem alguns que podem influenciar negativamente na fisiologia reprodutiva do touro, e, dessa forma, reduzir a fertilidade desses animais e causar esterilidade, tais como os fatores diretamente relacionados ao manejo e a nutrição. Dessa forma, torna-se de fundamental importância o conhecimento da fisiologia do desenvolvimento testicular e ponderal, e de como alguns fatores, como o manejo e o clima, podem interferir nestes processos. Portanto, o objetivo desta revisão é demonstrar como alguns fatores do ambiente, tais como o manejo e o clima, podem influenciar nessas características, com vistas à produção animal.(AU)

Knowledge of the physiology and weight of the testicular development, sexual precocity, ability and quality of sperm production, and the factors that potentially interfere with these processes are important for predicting the reproductive capacity of bulls. Among these factors, there are some that can negatively influence their reproductive physiology, and thus reduce their fertility, causing sterility, such as factors directly related to the management and nutrition. Thus, it is of fundamental importance to study the physiology of testicular development and weight, and how certain factors, such as management and climate, can interfere with these processes. Therefore, the aim of this review is to show how some environmental factors, such as management and climate, can influence these characteristics, aiming to animal production.(AU)

El conocimiento de la fisiología del desarrollo testicular y ponderal, la precocidad sexual, la capacidad de producción y la calidad del esperma, además de los factores que potencialmente interfieren en estos procesos, son importantes para predecir la capacidad reproductiva de los toros. Entre estos factores, hay algunos que pueden influir negativamente en la fisiología reproductiva del toro, y por lo tanto reducir la fertilidad de esos animales y causar esterilidad, tales como los factores directamente relacionados al manejo y la nutrición. Por lo tanto, es de fundamental importancia el conocimiento de la fisiología del desarrollo testicular y ponderal, y cómo ciertos factores, como el manejo y el clima, pueden interferir en estos procesos. Así, el objetivo de esta revisión es demostrar cómo algunos factores ambientales, tales como manejo y el clima, pueden influenciar en esas características, con miras a la producción animal.(AU)

Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II / Perfil de expressão de RNAm de enzimas esteroidogênicas e produção de esteroides a partir de células da teca bovina cultivadas in vitro e estimuladas por Angiotensina II

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells.(AU)

O objetivo deste trabalho foi verificar o efeito da Angiotensina II (Ang II) sobre a esteroidogenese nas células da teca bovina, cultivadas in vitro. Para isso, células da teca bovina foram obtidas de folículos maiores que 10 mm de diâmetro de ovários oriundos de abatedouro e submetidas a diferentes tratamentos em uma sequência de experimentos. No experimento 1, o perfil de expressão do RNAm de CYP17A1 foi avaliado nas células da teca em resposta ao LH (10ng ml-1) e/ou Ang II (0,1µM) em diferentes momentos de tratamento. No experimento 2, foi investigado o efeito dose-resposta de Ang II (0,001; 0,1 e 10µM), acrescido de insulina (100ng ml-1) e LH (100ng ̸ml) sobre a esteroidogênese nas células da teca bovina. O experimento 3 explorou os possíveis efeitos da Ang II por meio do tratamento de células da teca com saralasina (antagonista dos receptores da Ang II). Após 24 horas, nos experimentos 2 e 3, o meio de cultura foi coletado e avaliado quanto aos níveis de testosterona e androstenediona pela técnica de HPLC. Em paralelo, a expressão gênica de enzimas-chave da esteroidogênese (HSD3B2, CYP11A1, CYP17A1) e STAR foi avaliada por qRT-PCR. Não se observou diferença na produção de testosterona e androstenediona entre controle e grupos tratados, bem como, na expressão do RNAm para os genes estudados. Em conclusão, nossos resultados não demonstraram um papel da Ang II sobre a esteroidogenese nas células da teca bovina.(AU)

INSULINA AFETA O DESENVOLVIMENTO DE FOLÍCULOS PRÉ- ANTRAIS OVINOS CULTIVADOS IN SITU

A insulina pode desempenhar, no cultivo in vitro, efeitos sobre a foliculogênse os quais permitiram uma maior obtenção de folículos pré-antrais para posterior produção de embrião. Neste contexto, o objetivo deste trabalho foi determinar o efeito do hormônio insulina sobre o cultivo in vitro de fragmentos ovarianos da espécie ovina, associando ou não com FSH. Para ovários foram coletados em matadouros e armazenados em meio MEM-HEPES a 4 °C e levados ao laboratório. Para o cultivo in vitro, foram obtidos fragmentos do córtex ovariano (3x3x2mm) e cultivados em meio -MEM+ por 8 dias a 39°C com 5% de CO2. Após os cultivos os fragmentos foram processado e analisados na histológica para avaliação morfológica do desenvolvimento folicular (ativação, manutenção da morfologia e diâmetro do oócito e folículo), análise imuno-histoquímica para localização do antígeno de proliferação nuclear e expressão gênica do RNA mensageiro mRNA para o gene apoptótico (caspase 3) e da esteroidogênese (CYP17 e CYP19). O fluxo de informação permitiu separar o estudo em dois experimentos. Experimento 1: Cultivo in vitro dos tecidos, avaliando os efeitos do hormônio insulina nas concentrações de (0, 1, 10, 100, 1.000 e 10.000 ng/mL), sobre a ativação, desenvolvimento e manutenção da morfologia. Experimento 2: Cultivo in vitro com hormônio insulina (0, 1, 100 ng/mL) associada com FSH sobre a ativação, desenvolvimento, manutenção da morfologia, antígeno de proliferação nuclear (PCNA) e expressão dos genes caspase 3, CYP17 e CYP19. No experimento 1 os resultados demonstraram que: Os maiores percentuais de ativação foram observados nas concentrações de 1 e 100 ng/mL do hormônio (P < 0,05), a porcentagem de folículos morfologicamente normais foi significativamente maior (P< 0,05) para os tecidos cultivados em 1 e 10 ng/mL de insulina comparado aos tecidos cultivados apenas em -MEM+. Com relação ao crescimento não foi observada diferença do diâmetro do oócito e folículo após o cultivo in vitro em nenhuma das concentrações da insulina. Entretanto na concentração de 10,000 ng/mL houve uma redução significativa no diâmetro folicular quando comparado aos tecidos cultivados apenas em -MEM+ (P < 0,05). No experimento 2: Os resultados demonstraram que após 8 dias de cultivo com as concentrações de insulina (1, 10 e 100 ng/mL) associado com FSH, apresentaram uma maior ativação (P < 0,05). Do mesmo modo, a porcentagem de folículos morfologicamente normais foi significativamente maior na concentração de 10 ng/mL com e sem FSH (P < 0,05). A marcação do PCNA pela imunohistoquímica demonstrou a presença do antígeno no oócito, em todos os grupos analisados, e nas células da granulosa, apenas no grupo que não foi submetido ao cultivo. A porcentagem de pixel para a marcação do PCNA em todas as concentrações de insulina associada ao FSH, foi maiores que os grupos sem FSH (P<0,05). A expressão dos genes CYP17 e CYP19 foram identificada em todos os grupos, entretanto sem diferença significativa. A expressão do gene caspase 3 no grupo IN1FSH (P < 0,05) do que nos grupos IN10, IN100, IN10FSH e IN100FSH. Em conclusão a insulina no cultivo in vitro de fragmentos ovarianos de ovelhas, sozinha ou associada com FSH atua de forma dose-dependente na ativação folicular, preservando a morfologia, mas sem alterar o diâmetro folicular. Em alta concentração (10.000 ng/mL) a insulina possui efeito inibitório sobre o crescimento inicial dos folículos. Conclui-se, ainda, que a insulina pode reduzir a expressão do gene relacionado com a apoptose (caspase 3), quando em concentrações intermediárias (10 e 100 ng/mL) especialmente associados ao FSH.

Insulin may have effects on folliculogenesis in in vitro culture, which allowed a greater preantral follicles to be obtained for later embryo production. In this context, the objective of this work was to determine the effect of the hormone insulin on the in vitro culture of ovarian fragments of the ovine species, associated or not with FSH. For ovaries were collected in slaughterhouses and stored in MEM-HEPES medium at 4 ° C and taken to the laboratory. For in vitro culture, fragments of the ovarian cortex (3x3x2mm) and cultured in -MEM + medium were obtained for 8 days at 39 ° C with 5% CO2. After culture, the fragments were processed and histologically analyzed for morphological evaluation of follicular development (activation, maintenance of the morphology and diameter of the oocyte and follicle), immunohistochemical analysis for localization of the nuclear proliferation antigen and gene expression of mRNA messenger RNA for the apoptotic gene (caspase 3) and steroidogenesis (CYP17 and CYP19). The flow of information allowed to separate the study in two experiments. Experiment 1: In vitro culture of the tissues, evaluating the effects of the hormone insulin at the concentrations of (0, 1, 10, 100, 1.000 and 10.000 ng/mL) on the activation, development and maintenance of morphology. Experiment 2: In vitro culture with hormone insulin (0.1, 100 ng/mL) associated with FSH on the activation, development, maintenance of morphology, nuclear proliferation antigen (PCNA) and expression of caspase 3, CYP17 and CYP19 genes. In the experiment 1 the results showed that: The highest percentages of activation were observed at concentrations of 1 and 100 ng/mL of the hormone (P <0.05), the percentage of morphologically normal follicles was significantly higher (P <0.05) for tissues cultured in 1 and 10 ng / mL insulin compared to tissues cultured only in -MEM+. Regarding growth, no difference in oocyte and follicle diameter was observed after in vitro culture at any of the insulin concentrations. However, at the concentration of 10,000 ng / mL there was a significant reduction in follicular diameter when compared to tissues cultured only in -MEM+ (P <0.05). In the experiment 2, the results showed that after 8 days of culture with insulin concentrations (1, 10 and 100 ng / mL) associated with FSH, they presented a greater activation (P<0.05). Likewise, the percentage of morphologically normal follicles was significantly higher at 10 ng / mL concentration with and without FSH (P <0.05). The PCNA labeling by immunohistochemistry showed the presence of the antigen in the oocyte, in all the analyzed groups, and in the granulosa cells, only in the group that was not submitted to the culture. The percentage of pixel for PCNA labeling at all FSH associated insulin concentrations was higher than the non FSH groups (P <0.05). The expression of the CYP17 and CYP19 genes were identified in all groups, however without significant difference. The expression of the caspase 3 gene in the IN1FSH group (P <0.05) than in the IN10, IN100, IN10FSH and IN100FSH groups. In conclusion, insulin in the in vitro culture of ovarian sheep fragments alone or associated with FSH acts in a dosedependent manner on follicular activation, preserving the morphology, but without altering the follicular diameter. At high concentration (10,000 ng/mL) insulin has an inhibitory effect on the initial follicle growth. It is also concluded that insulin can reduce the expression of the gene related to apoptosis (caspase 3), when in intermediate concentrations (10 and 100 ng/mL) especially associated with FSH.

Influence of FSH on in vitro growth, steroidogenesis and DNA synthesis of buffalo (Bubalusbubalis) ovarian preantral follicles

The p resent study was designed to examine the effect of different doses of FSH on the growth , steroidogenesis and DNA synthesis of two different sizes of buffalo ovarian preantral follicles (PFs) during in vitro culture . Buffalo ovaries were collected from a local abattoir and PFs of small (150 μm) sizes were isolated through the microdissection method and cultured in TCM - 199 supplemented with 10% FB S, 1% ITS and 30 ng/ml EGF (control group) at 38.5 °C in 5% CO 2 with maximum humidity. The addition of three different doses of FSH (0.5, 1.0 and 2 .0 μg/ml) in the control medium served as the treatment groups . The culture medium was replenished every third day and spent culture medium was stored at - 30 °C for steroid hormone analysis. The results showed that the supplementation of FSH in the culture medium stimulated (P < 0.05) follicular growth , steroidogenesis and DNA synthesis compared to the control. The addition of FSH at 1.0 μg/ml in the culture medium stimulated better (P < 0.05) follicular growth, DNA synthesis and steroidogenesis compared to 0.5 μg/ml FSH , whereas a higher dose of FSH (2.0 μg/ml) inhibited follicular growth in both sizes of follicles . In conclusion, the present study demonstrate d that FSH plays a key role during the development of buffalo ovarian preantral follicles , as it stimulated i n vitro survival, growth rate, steroidogenesis and DNA synthesis of buffalo preantral follicle s at an optim al dose of 1.0 μg/ml.

Influence of FSH on in vitro growth, steroidogenesis and DNA synthesis of buffalo (Bubalusbubalis) ovarian preantral follicles

The p resent study was designed to examine the effect of different doses of FSH on the growth , steroidogenesis and DNA synthesis of two different sizes of buffalo ovarian preantral follicles (PFs) during in vitro culture . Buffalo ovaries were collected from a local abattoir and PFs of small (<150 μm) and large (>150 μm) sizes were isolated through the microdissection method and cultured in TCM - 199 supplemented with 10% FB S, 1% ITS and 30 ng/ml EGF (control group) at 38.5 °C in 5% CO 2 with maximum humidity. The addition of three different doses of FSH (0.5, 1.0 and 2 .0 μg/ml) in the control medium served as the treatment groups . The culture medium was replenished every third day and spent culture medium was stored at - 30 °C for steroid hormone analysis. The results showed that the supplementation of FSH in the culture medium stimulated (P < 0.05) follicular growth , steroidogenesis and DNA synthesis compared to the control. The addition of FSH at 1.0 μg/ml in the culture medium stimulated better (P < 0.05) follicular growth, DNA synthesis and steroidogenesis compared to 0.5 μg/ml FSH , whereas a higher dose of FSH (2.0 μg/ml) inhibited follicular growth in both sizes of follicles . In conclusion, the present study demonstrate d that FSH plays a key role during the development of buffalo ovarian preantral follicles , as it stimulated i n vitro survival, growth rate, steroidogenesis and DNA synthesis of buffalo preantral follicle s at an optim al dose of 1.0 μg/ml.(AU)

EXPRESSÃO DIFERENCIAL DO RECEPTOR DE LH, DA PROTEÍNA DE LIGAÇÃO DE MRNA DO LHR, BTA-MIR-222 E ENZIMAS ESTEROIDOGÊNICAS NO OVÁRIO BOVINO EM DESENVOLVIMENTO

EXPRESSÃO DIFERENCIAL DO RECEPTOR DE LH, DA PROTEÍNA DE LIGAÇÃO DE MRNA DO LHR, BTA-MIR-222 E ENZIMAS ESTEROIDOGÊNICAS NO OVÁRIO BOVINO EM DESENVOLVIMENTO Esteroides e gonadotrofinas são essenciais para a regulação do desenvolvimento folicular antral e os estágios finais do desenvolvimento pré-antral. Embora o receptor do hormônio luteinizante (LHR) tenha sido detectado nos folículos pré-antrais de ratos, coelhos e porcos, a expressão deste receptor no ovário fetal bovino não foi demonstrada. O presente estudo teve como objetivo quantificar a expressão do LHR e a abundância de mRNA da proteína de ligação LHR (LRBP), STAR, HSD3B1, CYP17A1 e CYP19A1 durante o desenvolvimento do ovário fetal bovino. Além disso, objetivamos identificar e quantificar a expressão de bta-miR-222 (microRNA regulador do gene LHCGR). Em resumo, a expressão de LHR foi observada no folículo pré-antral no ovário fetal de bovino e a abundância de mRNA foi menor no dia 150 do que no dia 60. No entanto, a abundância de mRNA da LRBP seguiu o padrão oposto. Semelhante a LRBP, a abundância de bta-miR-222 foi maior no dia 150 do que no dia 60 ou 90. Com relação à expressão gênica de enzimas esteroidogênicas; apenas a abundância de mRNA de STAR foi maior no dia 150 do que no dia 60. A proteína LHR foi detectada em oogônia, folículos primordiais, primários e secundários. Além disso, ambos os oócitos e células da granulosa apresentaram imunolocalização positiva para LHR. Em conclusão, estes resultados sugeriram o envolvimento da regulação do LHCGR / LBPB com mecanismos relacionados ao desenvolvimento de folículos pré-antrais, especialmente durante o estabelecimento de folículos secundários. Além disso, os presentes dados reforçaram que a expressão reduzida de mRNA de LHR em ovários fetais bovinos no dia 150 estava relacionada à maior expressão de LRBP e bta-miR-222.

Steroids and gonadotrophins are essential for the regulation of antral follicular development and the late stages of preantral development. Although the luteinizing hormone receptor (LHR) has been detected in the preantral follicles of rats, rabbits, and pigs, the expression of this receptor in bovine fetal ovary has not been demonstrated. The present study aimed to quantify the expression of the LHR and the mRNA abundance of the genes LHR binding protein (LRBP), STAR, HSD3B1, CYP17A1, and CYP19A1 during the development of bovine fetal ovary. In addition, we aimed to identify and quantify the expression of bta-miR-222 (a regulatory microRNA of the LHCGR gene). In summary, LHR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. The LHR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHR. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. With regard to the gene expression of steroidogenic enzymes; only the mRNA abundance of STAR was higher on day 150 than on day 60. In conclusion, these results suggested the involvement of LHCGR/LRBP regulation with mechanisms related to the development of preantral follicles, especially during the establishment of secondary follicles. Furthermore, the present data reinforced that the reduced expression of LHR mRNA in bovine fetal ovaries on day 150 was related to the higher expression of LRBP and bta-miR-222.

Papel da insulina sobre a esteroidogênese no corpo lúteo canino

Hipotetizamos que a insulina através de diferentes genes desempenhe um papel fundamental na regulação da esteroidogênese, e consequentemente nas funções das células luteínicas de cadelas não prenhes. Nosso objetivo in vivo foi mapear os genes diferencialmente expressos diretamente relacionados à cascata de sinalização insulínica e a esteroidogênese no CL canino, caracterizando sua expressão gênica e proteica no diestro. Além de caracterizar a via secundária de captação de glicose também decorrente da sinalização insulínica. Cadelas não gestantes foram submetidas à ovariosalpingohisterectomia a cada 10 dias entre os dias 10 e 60 (n=5/grupo) após a ovulação. Os CL coletados foram utilizados para sequenciamento de RNA (RNA-Seq) e validação por PCR em tempo real, e proteica por Western blotting e imunofluorescência. Nos experimentos in vitro, identificamos a diferença de respostas das células luteínicas após estímulo insulínico sob a expressão dos genes relacionados à esteroidogênese encontrados diferencialmente expressos, e ainda realizamos o bloqueio das vias PI3K, MAPK14 e MAPK1/2 para mensuração da produção de esteroides (n=4/grupo). Foram identificados sete genes diferencialmente expressos relacionados à sinalização insulínica: o IRS1, PI3KR3, PI3KCG, MAPK9, MAPK13 e MAPK14 e SOCS1, além de dois genes, CYP19A1 e HSD3B, identificados como envolvidos com a esteroidogênese. A expressão de CYP19A1 encontra-se associada positivamente com a expressão de IRS1 (r = 0,6169 P= 0,0326) e negativamente com a expressão de MAPK9 (r =-0,8851 P= 0,0001) e SOCS1 (r =-0,6628 P= 0,0188). Enquanto HSD3B encontra-se associado positivamente com MAPK9 (r = 0,7889 P=0,0621), PI3KR3 (r =0,9906 P=0,0001) e SOCS1 (r =0,9534 P=0,0032), e negativamente com IRS1 (r = -0,9555 P=0,0003) e MAPK14 (r =-0,9541 P=0,0032). A via secundária de captação de glicose mostrou que CAP1, CrKII apresentaram aumento de sua expressão no período em que ocorre o aumento da produção de P4, diferente de RAP e RHOQ que apresentaram menor expressão gênica nos dias 40, coincidindo com o aumento de E2 no diestro. Nos experimentos in vitro, sob estímulo insulínico, a expressão de CYP19A1 não apresentou diferença quando as células eram provenientes do dia 20 (P=0.0682; r=0.5745), diferentemente do dia 40 (P= <0.0001; r=0.9655), no qual houve aumento de sua expressão no grupo tratado com insulina em relação ao controle. Em relação à expressão de HSD3B, houve aumento de sua expressão no dia 20 (P=<0.0001; r=1.0) e 40 (P=0.0042; r=0.7134). A produção de P4 pelas células luteinicas cultivadas apresentou diminuição de sua expressão com o bloqueio de PI3K (P=0.0026; r=0.8671), MAPK14 (P=0.0009; r=0.9072) e MAPK1/2 (P=0.0407; r=0.6493), enquanto que a produção do E2 apresentou diminuição nos bloqueios com PI3K (P=0.0008; r=0.9676) e MAPK14 (P=<0.0001; r=0.9902), e não houve diferença de produção com o bloqueio de MAPK1/2 (P=0.0704; r=0.4639). Em conjunto, estes dados sugerem que MAPK e PI3K podem modular a esteroidogênese no CL canino, provavelmente via expressão de HSD3B e CYP19A1. A ativação da via CAP-CrKII-RHOQ-RAP, não esta envolvida neste processo. Concluimos que a insulina é capaz de modular a esteroidogênese no CL canino e que a resposta hormonal ao estímulo insulínico depende do dia do diestro.

We hypothesize that insulin plays a key role in steroidogenesis regulation and consequently the functions of luteal cells of non-pregnant female canines. In vivo goal was to map the differentially expressed genes directly related to the cascade of insulin signaling and steroidogenesis in the canine CL, characterizing its gene and protein expression along the diestrus. In addition, we intended to characterize the secondary pathway of glucose uptake also resulting from insulin signaling. Non-pregnant females were submitted to ovariosalpingohisterectomy every 10 days between days 10 and 60 (n=5/group) post- ovulation. The collected CL was used for RNA sequencing (RNA-Seq), validation by real-time PCR, and protein Western blotting and immunofluorescence. In in vitro experiments we identified different responses of luteal cells after insulin stimulation under the expression of differentially expressed steroidogenesis genes. We also performed PI3K, MAPK14 and MAPK1/ 2 pathway blockade to measure production of steroids (n = 4/group). Seven differentially expressed genes related to insulin signaling were identified: IRS1, PI3KR3, PI3KCG, MAPK9, MAPK13 and MAPK14 and SOCS1, in addition to two genes, CYP19A1 and HSD3B, identified as being involved with steroidogenesis. CYP19A1 expression is positively associated with the expression of IRS1 (r = 0.6169 P = 0.0326) and negatively with expressions of MAPK9 (r = -0.8851 P = 0.0001) and SOCS1 (r = -0.6628 P = 0.0188). HSD3B is positively associated with MAPK9 (r = 0.7889 P = 0.0621), PI3KR3 (r = 0.9906 P = 0.0001) and SOCS1 (r = 0.9534 P = 0.0032), and negatively with IRS1 (r = -0.9555 P = 0.0003) and MAPK14 (r = -0.9541 P = 0.0032). The secondary glucose uptake pathway showed that CAP1 and CrKII increased expression in the period of increased production of P4. RAP and RHOQ shows less gene expression on days 40, coinciding with the increase of E2, during diestrus. In in vitro experiments, under insulin stimulation, CYP19A1 expression did not alter in cells from day 20 (P = 0.0682; r = 0.5745). However, in cells from day 40 (P = <0.0001; r = 0.9655) expression was increased in the group treated with insulin in relation to the control. HSD3B expression increased on days 20 (P = <0.0001; r = 1.0) and 40 (P = 0.0042; r = 0.7134). The production of P4 by cultured luteal cells decreased expression with PI3K (P = 0.0026, r = 0.8671), MAPK14 (P = 0.0009, r = 0.9072) and MAPK1 / 2 (P = 0.0407, r = 0.6493 ). However, production of E2 decreased with PI3K (P = 0.0008; r = 0.9676) and MAPK14 (P = <0.0001; r = 0.9902) blockade, and did not alter with MAPK1/2 blockade (P = 0.0704, r = 0.4639). This data suggests that MAPK and PI3K can modulate steroidogenesis in canine CL, probably via HSD3B and CYP19A1 expression, and that activation of the CAP-CrKII-RHOQ-RAP pathway is not involved in this process. We conclude that insulin can modulate steroidogenesis in canine CL and that hormonal response to insulin stimulus depends on the day of the diestrus.

Involvement of vasoactive intestinal peptide (VIP) on ovarian physiology

In addition to the central nervous system, vasoactive intestinal peptide (VIP) containing nerves have been described throughout the female genital tract. VIP is reported to be produced by nerves fibers innervating follicles at all stages of development in rodents. There is growing evidence that VIP and their receptors play important roles in the local regulation of ovarian physiology mostly through cAMP pathway. It has been reported that VIP regulates the ovarian follicle survival and growth, oocyte maturation, ovulation and steroidogenesis. Studies also demonstrated that VIP inhibits apoptosis of rat follicles and is an important factor for the growth of preantral follicles enclosed in caprine ovarian tissue. Even though the addition of VIP to the culture medium did not improve in vitro maturation and fertilization of oocytes, it has been shown to stimulate ovulation in perfused rat ovaries. VIP is also involved in the regulation of steroidogenic activity. Therefore, this review aims to summarize current data on the importance of VIP on ovarian physiology.

Involvement of vasoactive intestinal peptide (VIP) on ovarian physiology

In addition to the central nervous system, vasoactive intestinal peptide (VIP) containing nerves have been described throughout the female genital tract. VIP is reported to be produced by nerves fibers innervating follicles at all stages of development in rodents. There is growing evidence that VIP and their receptors play important roles in the local regulation of ovarian physiology mostly through cAMP pathway. It has been reported that VIP regulates the ovarian follicle survival and growth, oocyte maturation, ovulation and steroidogenesis. Studies also demonstrated that VIP inhibits apoptosis of rat follicles and is an important factor for the growth of preantral follicles enclosed in caprine ovarian tissue. Even though the addition of VIP to the culture medium did not improve in vitro maturation and fertilization of oocytes, it has been shown to stimulate ovulation in perfused rat ovaries. VIP is also involved in the regulation of steroidogenic activity. Therefore, this review aims to summarize current data on the importance of VIP on ovarian physiology.(AU)

Aspectos morfológicos, vasculares e endócrinos de prenhezes produzidas por técnicas de reprodução assistida em bovinos

Perdas embrionárias e alterações gestacionais são frequentemente observadas em prenhezes de embriões bovinos manipulados in vitro. Sabe-se que tais anormalidades são resultantes de alterações epigenéticas ocasionadas pela manipulação dos gametas e/ou do embrião durante as técnicas de reprodução assistida (ARTs), com destaque para as técnicas de fecundação in vitro (FIV) e da clonagem por transferência nuclear de células somáticas (SCNT). Tais alterações resultam em distúrbios no desenvolvimento do concepto em algum momento crítico entre a fertilização e o parto, fornecendo bons modelos de estudos sobre a fisiopatologia de perdas embrionárias e dos distúrbios de desenvolvimento. Caracterizam-se como momentos críticos após a transferência do embrião (TE) o desenvolvimento embrionário no útero, o reconhecimento materno da gestação, a placentação e o desenvolvimento da placenta e do feto, os quais tem de ser transpassados sem nenhuma falha, permitindo um desenvolvimento normal do concepto até o termo. Sendo assim, o presente trabalho abordou três fases distintas do amplo período gestacional em prenhezes por ARTs. O Estudo 1 foi realizado durante o período peri-reconhecimento materno da gestação e objetivou descrever a abundância de expressão de genes estimulados pelo interferon tau (ISGs) de células mononucleares do sangue periférico (PBMCs) maternas em gestações oriundas de ARTs no primeiro mês de gestação; o Estudo 2 compreendeu os primeiros 35 dias de gestação e objetivou descrever as mudanças morfológicas e vasculares do complexo útero-concepto-ovário e o estímulo à expressão de ISGs em PBMCs em gestações de conceptos clonados por SCNT com diferentes fenótipos de desenvolvimento, sendo esses denominados gestação anembrionada e CL persistente; e o Estudo 3 foi conduzido durante o período pré-parto e objetivou descrever as alterações na produção de esteroides sexuais e corticosteroides em gestações produzidas por ARTs. Três hipóteses foram testadas: (1) Gestações de conceptos clonados por SCNT apresentam uma baixa e mais tardia estimulação de ISGs em PBMCs maternas quando comparadas com gestações de conceptos produzidos por FIV e IA; (2) O concepto clonado por SCNT apresenta um menor estímulo sobre mudanças morfológicas e vasculares do complexo útero-ovário e ISGs em PBMCs maternas durante os primeiros 35 dias de gestação, quando comparado com conceptos oriundos de IA; e (3) Gestações de embriões oriundos de ARTs apresentam alterações na dinâmica esteroidogênica no pré-parto quando comparados com gestações de IA. No estudo 1 foram coletadas amostras de sangue de gestações produzidas por inseminação artificial (IA), FIV e clonagem por SCNT, nos dias 15, 18, 20, 22, 24, 26, 28 e 31 pós-ovulação e foi realizada mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 2, gestações produzidas por IA e clonagem por SCNT, foram submetidas a escaneamentos ultrassonográficos dos ovários, útero e concepto a cada 3 dias do dia 14 ao 35 (dia 0 = ovulação) e amostras de sangue foram coletadas nos dias 15, 18, 20, 22, 24, 26, 28 e 31 para mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 3, foram coletadas amostras de sangue no último mês em gestações naturais, oriundas de FIV e de clonagem por SCNT para análise hormonal de 10 esteroides utilizando o método de espectrometria de massas multi-hormonal de alta resolução LC-MS/MS. O primeiro estudo mostrou semelhanças na expressão de genes estimulados pelo IFNT em gestações oriundas de ARTs e produzidas por IA. Entretanto, a estimulação nas gestações oriundas de ARTs aparentou ser quatro dias mais prolongada, sugerindo uma maior funcionalidade do trofectoderma em conceptos oriundos de ARTs. O segundo estudo demonstrou um aumento na expressão de ISGs em PBMCs maternas tanto em gestações de conceptos normais quanto em anormais, justificando a manutenção da função luteal mesmo na ausência de detecção do concepto por ultrasonografia. No terceiro estudo, demonstrou-se alterações na esteroidogênese nas gestações de embriões FIV e clonados no último mês de gestação, sendo essas compatíveis com a hiperativação da enzima aromatase durante todo o último mês de gestações oriundas de FIV e hiperativação das enzimas P450C11 e P450C21 trinta dias antes do parto em gestações oriundas de clonagem por SCNT. O presente estudo concluiu que conceptos oriundos de FIV e clonagem por SCNT apresentam um prolongamento no estímulo de ISGs pelo IFNT, conceptos clonados anormalos apresentam estímulo de ISGs, o que justifica a manutenção da função luteal, e, por fim, a cascata esteroidonênica que culmina com o parto apresenta-se alterada em gestações oriundas de FIV e clonagem por SCNT.

Pregnancy losses and gestational abnormalities are frequently observed in pregnancies from in vitro produced embryos in bovines. It is known that these abnormalities are due to epigenetic changes from the manipulation of gametes and/or embryo during the use of assisted reproduction techniques (ARTs), especially for the in vitro fertilization (IFV) and cloning by somatic cells nuclear transfer (SCNT). These changes results in disturbances of conceptus development in any critical stage between the fertilization and parturition, which provides good models for the study of physiopathology of embryo losses and disturbances of development. Critical stages after the embryo transfer (ET) to the uterus are characterized as the maternal recognition of pregnancy, placentation, and fetal-placental development, which needs to be surpassed without failures, in order to develop a normal conceptus until term. Therefore, the present work approached three distinct phases of the wide gestational period in pregnancies from ARTs. The Study 1 was conducted during the maternal peri-recongnition of pregnancy period and aimed to describe the expression of interferon stimulated genes (ISGs) in maternal peripheral blood mononuclear cells (PBMCs) in pregnancies derived of ARTs; the Study 2 comprise the first 35 days of pregnancy and aimed to describe morphological and vascular changes of the complex uterus-ovaries-conceptus, as well as the expression of ISGs in maternal PBMCs in pregnancies of conceptus cloned by SCNT with different phenotypes of development, denominated as anembryonic gestation and persistent CL; the Study 3 was conducted during the pre-partum period and aimed to describe changes in the production of sexual steroids and corticosteroids during the last month of pregnancies derived of ARTs. Three hypothesis were tested: (1) Pregnancies of conceptus cloned by SCNT presented a decrease and delay in the stimulation of ISGs in maternal PBMCs when compared with conceptuses produced by IFV and AI; (2) Stimulus from the conceptus for changes in the morphology and vasculature of the the uterus-ovarian complex, detected by ultrasonography in B and Doppler modes, and the stimulation of ISGs in maternal PBMCs during the first 35 days of pregnancy of conceptus cloned by SCNT are less intense when compared with conceptus derived from AI; and (3) Pregnancies derived of ARTs present changes in the steroidogenic dynamics in the pre-partum, when compared with pregnancies derived from AI. In Study 1 blood samples were collected from pregnancies produced by AI, IVF, and cloning by SCNT, at days 15, 18, 20, 22, 24, 26, 28, and 31 post-ovulation for the measurement of abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 2, pregnancies derived of AI and cloning by SCNT, were submitted to ultrasonographic scans for the evaluation and description of morphological and vascular changes in ovaries, uterus, and conceptus every 3 days from day 14 to 35 (day 0 = ovulation) and blood samples were collected on days 15, 18, 20, 22, 24, 26, 28 e 31 for the measurement of the abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 3, blood samples were collected during the last month of pregnancies naturally conceived, derived of IVF, and cloned by SCNT for the analysis of 10 steroids using the method of mass spectrometry high resolution LC-MS/MS. The first study showed similarities in the ISGs expression stimulation in pregnancies derived of ARTs and AI. However, the stimulation in the ART derived pregnancies was apparently 4 days longer, suggesting a greater placental function in conceptus derived of ARTs. The second study showed an increase in ISG expression in both normal and abnormal conceptus development, which justifies the maintenance of CL in the absence of a conceptus structure detected by ultrasonography. In the third study, was detected changes in the steroidogenesis of pregnancies derived of IFV and cloning by SCNT during the last month of pregnancy, which are compatible with the hyperactivation of the aromatase enzyme during the last month of IFV derived pregnancies, and hyperactivation of the enzymes P450C11 and P450C21 thirty days before parturition in pregnancies derived of cloning by SCNT. The present study concludes that conceptus derived of IFV and cloning by SCNT present a prolonged stimulus of ISGs, cloned conceptus with anomalous development presents a stimulus of ISGs, which justifies the CL function maintenance, and, ultimately, the steroidogenic cascade that culminates with the term is altered in pregnancies derived from IFV and cloning by SCNT.