RESUMO

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

Assuntos

RESUMO

Failure of ovulation can lead to follicular persistence, one of the main components of the pathogenesis of cystic ovarian disease (COD) in dairy cattle. Follicular persistence causes the permanence of a functional follicular structure in the ovary, which alters the cyclicity of the female and causes infertility. The aim of the present study was to evaluate the expression of estrogen receptors (ESR) 1 and 2, and the coregulatory proteins NCOA1, NRIP1 and LCOR by immunohistochemistry, in antral and preovulatory/persistent follicles in a model of follicular persistence induced by low levels of progesterone, to detect incipient changes during COD development, on the expected day of ovulation (P0) and after 5 (P5), 10 (P10) and 15 (P15) days of follicular persistence. Twenty-five Holstein cows were used, which were distributed in 5 groups: control group (n = 5), group P0 (n = 5), group P5 (n = 5), group P10 (n = 5), group P15 (n = 5). ESR1 expression was lower in antral follicles of the P5 (theca), P10 and P15 (theca and granulosa) groups relative to the control group (p < 0.05), and also lower in granulosa cells of persistent follicles of the P5, P10 and P15 groups than in dominant follicles of the control group (p < 0.05), without differences in theca cells. ESR2 expression showed no differences between groups. The ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as from 5 days of persistence (p < 0.05). NCOA1 expression was higher in granulosa cells of both antral and persistent follicles from the P0 group relative to the P5 and P10 groups, but showed no differences with the control and P15 groups (p < 0.05). Theca cells of antral and persistent follicles showed higher expression in the P0 and P15 groups in relation to the control, P5 and P10 groups (p < 0.05). No differences were detected for NRIP1 in antral, dominant and persistent follicles between groups. LCOR expression showed a decrease in granulosa cells of antral follicles from all persistence groups relative to the control group (p < 0.05). In theca cells, antral follicles of the P10 group showed lower LCOR expression than the control group (p < 0.05). LCOR expression was similar for dominant and persistent follicles. Considering that the ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as well as the decreased LCOR and NCOA1 expression, we may assume that, at the early stages of persistence, there is a negative regulation of ESR transcription. This coincides with the effects of estrogens through ESR on proliferation and apoptosis among other processes that favor follicular persistence. The results obtained provide relevant information in the knowledge of local events during the development of follicular persistence that could explain the failures in the reversion of the disease through hormonal treatments and the high recurrence rates reported for COD. In addition, it contributes to the study and identification of possible therapeutic targets, for the design of new treatments.

Assuntos

RESUMO

The objective of this study was to describe a case of a granulosa cell tumour (GCT) of incipient formation and to characterize it by its immunohistochemical pattern and hormonal profile. The case presented corresponds to a 7-year-old Holstein cow without reproductive disorders. No alterations were observed at rectal palpation, neither in the ultrasonography nor in the hormonal profile. A GCT concomitant with normal follicular development was diagnosed. Through a panel of immunohistochemical markers, a highly differentiated pattern could be determined in the GCT, which preserves the expression of steroid receptors (ESR1, ESR2 and PR) typical of granulosa cells, but does not express the enzymes for the synthesis of androgens (CYP17A1) and oestrogens (CYP19A1). In addition, the expression of co-regulators of steroid hormone receptors and neuroendocrine markers was described for the first time in a GCT in cattle. These results increase the information about GCTs in cattle before the ovarian function is compromised.

Assuntos

RESUMO

Resumen El descubrimiento de un nuevo principio activo farmacéutico implica estudios preclínicos, que tienen como objetivo demostrar que es eficaz y seguro para un posterior ensayo en seres humanos. Esto conduce a la necesidad de desarrollar tecnologías que aprovechen las nuevas herramientas analíticas disponi bles dentro de un contexto donde los resultados de las pruebas realizadas, estén plenamente documentados, bajo sistemas de buenas prácticas de laboratorio auditables. En esta revisión se actualizan y describen algunos de los ensayos realizados en la etapa preclínica del desarrollo de un nuevo fármaco y el estado actual de la tecnología analítica empleada para el dosaje de diferentes biomarcadores sanguíneos de interés. Se analizaron los biomarcadores más relevantes, las normativas de validación de las técnicas analíticas empleadas para su determinación y los problemas que se presentan al tratar de aplicarlas.

Abstract New drug discovery involves preclinical studies to demonstrate its effectivity and safety for further tests in humans. This leads to the need to develop technologies that take advantage of the new analytical tools available within a context where the results of the tests carried out are fully documented, under auditable systems of good laboratory practice. This review updates and describes some of the tests carried out in the preclinical stage of the development of a new drug and the current state of the analytical technology used to measure different blood biomarkers of interest. Biomarker parameters were analyzed at the physiological level, considering both the validation regulations of the analytical techniques used for their determination as the problems that arise when trying to apply them, since many of these biomarkers are endogenous compounds in the used matrices.

Assuntos

RESUMO

New drug discovery involves preclinical studies to demonstrate its effectivity and safety for further tests in humans. This leads to the need to develop technologies that take advantage of the new analytical tools available within a context where the results of the tests carried out are fully documented, under auditable systems of good laboratory practice. This review updates and describes some of the tests carried out in the preclinical stage of the development of a new drug and the current state of the analytical technology used to measure different blood biomarkers of interest. Biomarker parameters were analyzed at the physiological level, considering both the validation regulations of the analytical techniques used for their determination as the problems that arise when trying to apply them, since many of these biomarkers are endogenous compounds in the used matrices.

El descubrimiento de un nuevo principio activo farmacéutico implica estudios preclínicos, que tienen como objetivo demostrar que es eficaz y seguro para un posterior ensayo en seres humanos. Esto conduce a la necesidad de desarrollar tecnologías que aprovechen las nuevas herramientas analíticas disponibles dentro de un contexto donde los resultados de las pruebas realizadas, estén plenamente documentados, bajo sistemas de buenas prácticas de laboratorio auditables. En esta revisión se actualizan y describen algunos de los ensayos realizados en la etapa preclínica del desarrollo de un nuevo fármaco y el estado actual de la tecnología analítica empleada para el dosaje de diferentes biomarcadores sanguíneos de interés. Se analizaron los biomarcadores más relevantes, las normativas de validación de las técnicas analíticas empleadas para su determinación y los problemas que se presentan al tratar de aplicarlas.

Assuntos

RESUMO

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1ß, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1ß, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1ß and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1ß and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Assuntos

RESUMO

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH-induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH-induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH-induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level.

Assuntos

RESUMO

The objective of this work was to evaluate proliferation and apoptosis in the bovine ovary in a model of follicular persistence induced by low levels of progesterone to detect incipient changes during cystic ovarian disease development on the expected day of ovulation (day 0) and after 5, 10, and 15 days of follicular persistence. We analyzed cell proliferation by evaluating the expression of Ki-67 and apoptosis by evaluating caspase-3, BAX, and BCL2 expression. Proliferation was similar in the granulosa and theca cells of antral follicles in the P0 group (treated with progesterone up to the expected day of ovulation) and in the control group. A decrease in cell proliferation was detected after 5 days of persistence (P5) in relation to P0 (p < 0.05). Similar changes were found in the granulosa cells of the persistent follicles in relation to the control group (p < 0.05). Caspase-3 expression was similar in granulosa cells of antral follicles at early stages of persistence, with an increase after 15 days of persistence (p < 0.05). In the granulosa cells of group P10 (10 days of persistence), caspase-3 expression was reduced relative to that of antral follicles from the control group (p < 0.05). BCL2 expression was higher in granulosa cells of the persistent follicles of group P0 relative to the control follicles, with no changes in BAX expression, which was increased in persistent follicles of group P15 (p < 0.05). Similar results were observed in theca cells at initial stages of persistence. The results show that, initially, proliferation is maintained with low apoptosis and an increase in cell survival.

Assuntos

RESUMO

Ovarian cystic follicles are the sign of important causes of reproductive failure in numerous species. In this review, some morphological, endocrinological and clinical aspects of cystic follicles in women, cows, mares, sows and bitches are discussed. Follicular cysts are the consequence of the failure of a mature follicle to ovulate at the appointed time of ovulation in the estrous cycle. Although the etiology of follicular cysts remains unknown, this review examines the evidence about the role of endocrine signaling systems in the specific disease or syndrome in each of the species mentioned above. This review also describes, the changes in the pathways of endocrine mechanisms that would trigger disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. The knowledge of the morphological and endocrinological nature of cystic follicles in different species can provide relevant information to better understand specific diseases when it is integrally analyzed from the comparative medicine viewpoint.

Assuntos