ACTH impairs the migratory and secretory profile of mononuclear cells during proestrus in cattle.

The aim was to evaluate the effect of ACTH on the mechanisms involved in peripheral blood mononuclear cells (PBMCs) infiltration into the ovary during dairy cattle proestrus. Regarding this, proper expression pattern of adhesion molecules must take place both in PBMCs and in endothelial cells. Argentinian Holstein cows (n = 12) were treated with 100 IU of ACTH every 12 h for 4 days before ovulation when ovariectomy was performed (day 18). Blood samples were taken on day 15 (0 h) and immediately before (72 h) and after (74 h) the last ACTH administration. In PBMCs, flow cytometry was performed to analyze CD44, CD11b and CD62-L expression along with gene expression of chemokines' receptors. Interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production was analyzed by flow cytometry after exposing PBMCs to autologous follicular fluid. In ovarian blood vessels, expression of the vascular endothelium cell adhesion-1 (VCAM-1) and the platelet endothelial cell adhesion molecule-1 was evaluated by immunohistochemistry. In T-lymphocytes, the expression of CD44 and CD11b was lower at 72 h in ACTH-treated cows (P < 0.05). In monocytes, the expression of CD11b and CD62-L was lower at 72 h in ACTH-treated cows (P < 0.05). Also, the percentage of IL-4+ cells was higher in ACTH-treated cows, meanwhile, the percentage TNF-α+ cells was lower in ACTH-treated cows (P < 0.05). Finally, in the vessels associated with the preovulatory follicle VCAM-1 immunoexpression was lower in ACTH-treated cows (P < 0.05). Here, we present novel insights into the effect of stress during the preovulatory period on the inflammatory pathway necessary for ovulation.

Endometrial expression of members of the IL-1 family: their involvement in delayed conception of dairy cows.

The cytokines of the interleukin-1 (IL-1) family are closely involved in the resolution of inflammation in cows with metritis and endometritis. However, little is known about the role of these cytokines beyond uterine regression in the absence of disease, especially around conception. Thus, the aim of this study was to examine the gene and protein expression of IL-1α, IL-1ß, IL-1RI, IL-1RII and IL-1Ra in endometrial biopsies previous to conception, to evaluate the possible association of these cytokines with delayed conception in dairy cows. Gene and protein expression levels were evaluated by real-time PCR and immunohistochemistry, respectively. The gene expression levels of cytokines were not associated with the duration of the period to conception following parturition. However, high protein expression of IL-1ß and low protein expression of IL-1Ra were significantly associated with early conception. These results suggest that an imbalanced protein expression of IL-1ß and IL-1Ra in the endometrium of dairy cows could be part of the maternal immune response mechanism necessary to propitiate early conception and probably to maintain pregnancy.

Contribution of key elements of nutritional metabolism to the development of cystic ovarian disease in dairy cattle.

The alteration of signaling molecules involved in the general metabolism of animals can negatively influence reproduction. In dairy cattle, the development of follicular cysts and the subsequent appearance of ovarian cystic disease (COD) often lead to decreased reproductive efficiency in the herd. The objective of this review is to summarize the contribution of relevant metabolic and nutritional sensors to the development of COD in dairy cows. In particular, we focus on the study of alterations of the insulin signaling pathway, adiponectin, and other sensors and metabolites relevant to ovarian functionality, which may be related to the development of follicular persistence and follicular formation of cysts in dairy cattle. The results of these studies support the hypothesis that systemic factors could alter the local scenario in the follicle, generating an adverse microenvironment for the resumption of ovarian activity and possibly leading to the persistence of follicles and to the development and recurrence of COD.

A review on inflammation and angiogenesis as key mechanisms involved in the pathogenesis of bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD.

Hemodynamic changes detected by Doppler ultrasonography in the ovaries of cattle during early development of cystic ovarian disease.

A common reproductive disease in dairy cattle is Cystic Ovarian Disease. To study its development, there was use of an experimental model of follicular persistence to detect hemodynamic changes occurring in ovaries by using Doppler ultrasonography. After estrous synchronization, control cows received no additional treatment and were evaluated at proestrus (CG), whereas treated cows (PG) received sub-luteal doses of progesterone for 15 days and were evaluated at proestrus, and after 0, 5, 10 and 15 days of follicular persistence. Spectral Doppler was used to evaluate blood flow in the ovarian artery, and power Doppler for evaluation of blood flow in the ovarian parenchyma and follicular wall of persistent and dominant preovulatory follicles. Findings using power Doppler signals indicated there were no differences between groups in the parenchyma of both right (P =  0.455) and left (P =  0.762) ovaries. In contrast, power Doppler signals of blood flow were less in walls of persistent follicles from day 0 to 15 when there was follicular persistence than in dominant follicles of the CG (P <  0.001). Blood flow in ovarian arteries was less (P <  0.05) in diastolic velocity and time averaged maximum velocity in all PG groups than in the CG. Peak systolic velocity was less (P <  0.05) in all PG than in the CG, with the exception of P15 (P >  0.05). These findings indicate there are marked changes in blood irrigation area of walls of persistent follicles during the 15 days of follicular persistence.

Contribution of the VEGF system to the follicular persistence associated with bovine cystic ovaries.

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.

Alterations in key metabolic sensors involved in bovine cystic ovarian disease.

High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5' adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD.

Impaired insulin signaling pathways affect ovarian steroidogenesis in cows with COD.

Cystic ovarian disease (COD) represents an important cause of infertility in dairy cattle and is associated with multiple physiological disorders. Steroidogenesis, which is necessary to ensure normal ovarian functions, involves multiple enzymatic pathways coordinated by insulin and other proteins. We have previously shown that cows with COD have an altered insulin response. Therefore, in the present study, we evaluated further alterations in intermediates downstream of the PI3K pathway and pathways mediated by ERK as critical signals for the expression of steroidogenic enzymes in the ovaries of control cows and cows with spontaneous COD. To this end, we evaluated the gene and protein expression of pan-AKT, mTOR, ERK1/2, and steroidogenic enzymes by real-time PCR and immunohistochemistry. Steroid hormone concentrations were assessed at systemic and intrafollicular level. Results showed altered expression of intermediate molecules of the insulin signaling pathway, whose action might modify the synthetic pathway of steroidogenic hormones. Similarly, the expression of steroidogenic enzymes and the concentration of progesterone in serum and follicular fluid were altered. These alterations support the hypothesis that systemic factors contribute to the development and/or maintenance of COD, and that metabolic hormones within follicles such as insulin exert determinant effects on ovarian functionality in cows with COD.

Altered Expression of Anti-Müllerian Hormone during the Early Stage of Bovine Persistent Ovarian Follicles.

Anti-Müllerian hormone (AMH) is a homodimeric glycoprotein expressed exclusively in the gonads. This hormone is an important regulator of the early growth of follicles through inhibitory effects on the recruitment of primordial follicles into the pool of growing follicles and on granulosa cell proliferation. Cystic ovarian disease (COD) is an important disorder affecting the fertility of dairy cattle. In the present study, we evaluated the expression of AMH in granulosa cells and AMH secretion into follicular fluid in pre-ovulatory follicles from control cows, animals with spontaneously arising COD and during the development of the disease, at 5, 10 and 15 days of follicular persistence. To this end, after an oestrous synchronization protocol, low doses of progesterone was administered for 5, 10 and 15 days after the expected day of ovulation (day 0 of follicular persistence) in treated cows (groups P5, P10 and P15, respectively), using an intravaginal progesterone-releasing device. Results showed a decrease in the expression of AMH in granulosa cells throughout folliculogenesis (P <0.05) and in the spontaneously arising follicular cysts and persistent follicles related to the control group (P <0.05). There was also a higher concentration of AMH in the follicular fluid of persistent follicles at 10 and 15 days of follicular persistence (P <0.05). Together, these results may indicate an alteration in AMH expression and secretion, which occurs early in folliculogenesis and incipiently during the development of COD, and which could contribute to the recurrence of this disease in cattle.

Altered expression of IL-1ß, IL-1RI, IL-1RII, IL-1RA and IL-4 could contribute to anovulation and follicular persistence in cattle.

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. The main signs of this infertility are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1ß, IL-1RI, IL-1RII, IL-1RA and IL-4 in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged administration of progesterone in dairy cows. Protein expression of IL-1ß, IL-1RI, IL-1RII, IL-1RA and IL-4 was evaluated by immunohistochemistry. Additionally, IL-1ß and IL-4 concentrations in follicular fluid and serum were determined by ELISA. In granulosa cells, IL1-RII and IL-4 expression was higher in follicles with different persistence times than in the control dominant follicles. IL-1RA expression was higher in persistent follicles of the P15 group (15 days of follicular persistence) than in those of the control group. In theca cells, IL-1RII expression was higher in persistent follicles of the P0 group (expected time of ovulation) than in dominant follicles from the control group (p < .05) and the other persistence groups, whereas IL-4 expression was higher in persistent follicles of groups P0 and P15 than in the dominant follicles of the control group (p < .05). Differences between serum and follicular fluid within each group were detected only in P0 for IL-1ß, and in the control, P10 and P15 groups for IL-4 (p < .05). These results complement previous results, evidencing that early development of COD in cows is concurrent with an altered expression of cytokines in different ovarian follicular structures and may contribute to the follicular persistence and ovulation failure found in cattle with follicular cysts.

Role of Components of the Insulin-like Growth Factor System in the Early Stages of Ovarian Follicular Persistence in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. The initiation of the IGF response is a result of interactions between IGF-binding proteins (IGFBPs) and IGFBP proteases, mainly pregnancy-associated plasma protein A (PAPP-A). IGFBPs bind IGFs with high affinity and consequently regulate their access to IGF receptors (IGFRs). The aim of this research was to determine variations in components of the IGF system in the ovaries of cows with persistent follicles induced by long-term administration of progesterone. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes occur early in the development of COD. The concentrations of IGF1 and IGFBP4 in follicular fluid were similar in all groups with follicular persistence and in control antral follicles. IGFR1 and IGFBP4 expression in situ were higher in granulose cells in persistent follicles than in control follicles. No differences were found in PAPP-A concentration within follicular fluid in persistent follicles relative to control antral follicles. These data support the hypothesis that the IGF system is altered in the initial stages of development of follicular persistence and has a determinant role in ovarian function in cattle.

Altered expression of cytokines IL-1α, IL-6, IL-8 and TNF-α in bovine follicular persistence.

In dairy cattle, cystic ovarian disease (COD) is an important cause of subfertility, and two of the main signs are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1α, IL-6, IL-8 and TNF-α in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged treatment with progesterone in dairy cows. Protein expression of IL-1α, IL-6, IL-8 and TNF-α was evaluated by immunohistochemistry. Additionally, IL-6 concentration in follicular fluid and serum was determined by ELISA. IL-1α, IL-6, IL-8 and TNF-α expression was increased in follicles with different persistence times in relation to the control dominant follicles, in granulosa cells. For IL-6, IL-8 and TNF-α, this increase was detected early (P0: expected time of ovulation and/or P5: 5 days of follicular persistence). Additionally, theca cells showed an increase in IL-6 in antral (groups P10 and P15) and persistent follicles (group P10) related to dominant follicles from the control group (p < 0.05). Serum concentration of IL-6 was higher in groups P5, P10 and P15 than in control cows (p < 0.05). The results show evidence that early development of COD in cows is concurrent with altered expression of these cytokines in different ovarian follicular structures and may contribute to the follicular persistence and endocrine changes found in cattle with follicular cysts.

Involvement of Matrix Metalloproteinases and their Inhibitors in Bovine Cystic Ovarian Disease.

The most important regulators of tissue remodelling during ovarian follicular growth, development, ovulation and atresia are gonadotropins, steroid hormones, growth factors and different proteolytic enzymes. Matrix metalloproteinases (MMPs) such as collagenase or gelatinase (i.e. MMP-1, -8, -2 and -9) and associated tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4) control connective tissue remodelling during follicular rupture. In this study, we hypothesized that an imbalance in the MMP-TIMP system may be an intra-ovarian component that contributes to the pathogenesis of cystic ovarian disease (COD) in cows. Taking into account that the control of MMP activity by TIMPs could determine their effects in both physiological and pathological conditions, MMP and TIMP mRNA and protein expression was examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively, in ovaries from control cows and cows with COD. Expression of mRNA encoding MMP-2, TIMP-1 and TIMP-2 was lower in follicular cysts than in control pre-ovulatory follicles, while the results by IHC showed this imbalance only for TIMP-2 protein expression. Additional analysis by zymography to evaluate the gelatinase activity of MMP-2 and MMP-9 demonstrated higher MMP-2 activity in follicular fluid (FF) of cysts than in FF of pre-ovulatory follicles. On the other hand, MMP-9 activity was increased in follicular cysts and absent in the FF of pre-ovulatory follicles. These findings suggest that the altered mRNA expression and activities of the MMP-TIMP system may be related to the failure in ovulation and follicular development observed in COD.

Altered Expression of Pro-inflammatory Cytokines in Ovarian Follicles of Cows with Cystic Ovarian Disease.

A growing body of evidence suggests that ovulation shares many of the features of an inflammatory reaction and that cytokines play many diverse and important roles in reproductive biology. The aim of this study was to examine the expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6 and tumour necrosis factor (TNF)-α in ovarian cells from cows with cystic ovarian disease (COD) as compared with that in ovarian structures from regularly cycling cows. Expression of genes encoding IL-1α, IL-6 and TNF-α was detected by real-time polymerase chain reaction in follicular cells from ovaries from healthy cows and cows with COD with no significant differences. However, immunohistochemistry showed increased expression of IL-1α, IL-6 and TNF-α in cystic follicles, suggesting that this expression may be related to the persistence of follicular cysts. The effect of COD was evident for IL-1α and TNF-α, and a follicular structure-disease interaction was observed in the expression of all the cytokines evaluated. Thus, altered expression of these proinflammatory cytokines may be related to ovulation failure and development of follicular cysts.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

Identificação imuno-histoquímica de VEGF e IGF-1 em ovários de cadelas no anestro e estro / Immunohistochemical identification of VEGF and IGF-1 in ovaries of bitches on anestrus and estrus

Com o objetivo de verificar a presença de VEGF e IGF-1 nos ovários de cadelas, foram realizadas análises imuno-histoquímicas do estroma cortical; teca e granulosa de folículos secundários, terciários e terciários pré-ovulatórios luteinizados; e ovócitos de folículos primários, secundários e terciários de ovários de cinco cadelas em anestro (Anest) e cinco em estro (Est). A identificação das fases do ciclo estral foi realizada por citologia vaginal associada a dosagem plasmática de progesterona. Os ovários foram submetidos a tratamento imuno-histoquímico para identificação de VEGF (anticorpo primário PU 360-UP, Biogenex, USA; diluição 1:30) e IGF-1 (anticorpo primário PabCa, Gro-Pep, Austrália; diluição 1:100). Determinou-se um índice de imunomarcação (IM), para cada tecido avaliado, pela razão entre a área positivamente marcada dividida pela área total analisada. Para os ovócitos, verificou-se imunomarcação positiva ou negativa. As comparações de IM entre tecidos foram realizadas pelo teste de Wilcoxon (diferentes tecidos em mesmo grupo) ou Mann-Whitney (mesmo tecido entre diferentes grupos), todas no nível de 5% de significância. VEGF e IGF-1 foram identificados, de forma semelhante (P>0,05), em todas as estruturas avaliadas em ambos os grupos experimentais. Conclui-se que esses fatores de crescimento estão presentes em cadelas no anestro e estro, no estroma cortical ovariano, folículos em diferentes estádios de desenvolvimento e ovócitos.(AU)

In order to verify the presence of VEGF and IGF-1 in the ovaries of bitches, immunohistochemical analyzes of the cortical stroma; theca and granulosa of secondary, tertiary and tertiary luteinized preovulatory follicles; and oocytes of primary, secondary and tertiary follicles of ovaries from five bitches in anestrous (Anest) and five in estrus (Est) was performed. The identification of the phases of the estrous cycle was performed by vaginal cytology associated with the measurement of plasma progesterone. The ovaries were treated for immunohistochemical identification of VEGF (PU 360 primary antibody-UP, Biogenex, USA, dilution 1:30) and IGF-1 (primary antibody PabCa, Gro-Pep, Australia; 1:100 dilution). The immunostaining index (MI) was determined for each tissue by the ratio of positively marked area divided by total analyzed area. For oocytes immunostaining was determined as positive or negative. Comparisons of IM between tissues were performed with the Wilcoxon test (deferent tissues in the same group) or Mann-Whitney test (same tissue between different groups), all at 5% significance level. VEGF and IGF-1 have been similarly identified (P>0.05) in all structures evaluated in both groups. It is concluded that in bitches in estrus and anestrous these growth factors are present in ovary cortical stroma, follicles at different stages of development and oocytes.(AU)

Impaired insulin signaling pathway in ovarian follicles of cows with cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence.

Altered expression of transforming growth factor-beta isoforms in bovine cystic ovarian disease.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors may contribute to follicular persistence. Transforming growth factor-beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)-induced COD. In the oestrous-synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous-synchronized control group with that in ACTH-induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH-induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.

Ovarian localization of 11ß-hydroxysteroid dehydrogenase (11ßHSD): effects of ACTH stimulation and its relationship with bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and 11ßHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11ßHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11ßHSD1 in tertiary follicles and follicular cysts and a decrease in 11ßHSD2 in follicular cysts. Moderate expression of 11ßHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11ßHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11ßHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.

Interaction between IGF1 and IGFBPs in bovine cystic ovarian disease.

Cystic ovarian disease (COD) is one of the main factors responsible for reproductive disorders in cattle. Although the pathogenesis and mechanism of cyst formation are not fully understood, it has been proposed that the IGF system could play an essential role, as it is a key intraovarian regulator. The aim of the present study was to determine whether the altered levels in IGF1 detected in bovines with COD are associated with changes at mRNA level or with differential modulation by IGFBPs. The mRNA levels of the IGF components studied were analyzed by real time PCR and in situ hybridization, and IGFBP expression and activity were assayed by immunohistochemistry and ligand blot, respectively. Results showed a decreased IGF1 mRNA level due to a lower granulosa cell gene expression in cystic follicles (P<0.05). Results also showed variations in IGFBP expression in the intraovarian cellular compartment and concentration in follicular fluid, and suggest that IGFBP3 is a key regulator of intrafollicular IGF1 in animals with COD.