Bovine Cystic Ovarian Disease Research Articles

Follicular structures of cows with cystic ovarian disease present altered expression of cytokines.

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-1β, 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-1β, 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-1β 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-1β 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.

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.

Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinases as Plasma Indicators of Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle and has a high economic impact on farmers. COD is caused by an endocrine imbalance within the hypothalamic-pituitary-ovarian axis preventing the mature Graafian follicle from ovulating. The cause at the molecular level is not well understood. However, the matrix metalloproteinases (MMP) and plasminogen activator/plasmin families of extracellular (ECM) matrix-degrading proteinases are involved in Graafian follicle breakdown and oocyte release in the ovulation process. Our research investigated the possibility of using plasma concentrations of MMP-2 and -9 and their natural tissue inhibitors (TIMP) -1 and -2 as prognostic indicators of COD. Plasma samples from cystic and non-cystic dairy cows were analyzed using ELISA. Although plasma concentrations of MMP-2 and -9 were greater and TIMP-2 was lower in non-cystic compared to cystic cows, no significant differences were observed in MMP-2 and -9 and TIMP-1 and -2 due to cyst status. However, the TIMP-1:MMP-9 and TIMP-2:MMP-2 molar ratios were greater, (P = 0.099) and (P = 0.038), respectively, in cystic compared to non-cystic cows, suggesting a proteolytic insufficiency in cows with COD that may be a contributing factor to the anovulatory pathology. These data may provide the groundwork for future research and development of tools for dairy farmers to selectively choose replacement heifers less likely to develop COD.

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.

BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1.

Molecular aspects of bovine cystic ovarian disease pathogenesis.

Cystic ovarian disease (COD) is one of the main causes of reproductive failure in cattle and causes severe economic loss to the dairy farm industry because it increases both days open in the post partum period and replacement rates due to infertility. This disease is the consequence of the failure of a mature follicle to ovulate at the time of ovulation in the estrous cycle. This review examines the evidence for the role of altered steroid and gonadotropin signaling systems and the proliferation/apoptosis balance in the ovary with cystic structures. This evidence suggests that changes in the expression of ovarian molecular components associated with these cellular mechanisms could play a fundamental role in the pathogenesis of COD. The evidence also shows that gonadotropin receptor expression in bovine cystic follicles is altered, which suggests that changes in the signaling system of gonadotropins could play a fundamental role in the pathogenesis of conditions characterized by altered ovulation, such as COD. Ovaries from animals with COD exhibit a disrupted steroid receptor pattern with modifications in the expression of coregulatory proteins. These changes in the pathways of endocrine action would trigger the changes in proliferation and apoptosis underlying the aberrant persistence of follicular cysts. Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/R251/suppl/DC1.

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.

Role of activin, inhibin, and follistatin in the pathogenesis of bovine cystic ovarian disease

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Although many researchers have focused their work on the endocrine changes related to this disease, evidence indicates that intraovarian components play an important role in follicular persistence. Activin, inhibin, and follistatin participate as intraovarian regulatory molecules involved in follicular cell proliferation, differentiation, steroidogenesis, oocyte maturation, and corpus luteum function. Given the importance of these factors in folliculogenesis, we examined the expression and immunolocalization of activin/inhibin βA-subunit, inhibin α-subunit, and follistatin in the ovaries of healthy estrus-synchronized cows and in those of cows with spontaneous or adrenocorticotropic hormone (ACTH)-induced COD. We also studied inhibin B (α βB) levels in serum and follicular fluid. We found an increased expression of the βA-subunit of activin A/inhibin A, the α-subunit of inhibin, and follistatin in granulosa cells of spontaneous follicular cysts by immunohistochemistry, and decreased concentrations of inhibin B (α βB) in the follicular fluid of spontaneous follicular cysts. These results, together with those previously obtained, indicate that the expression of the components of the activin–inhibin–follistatin system is altered. This could lead to multiple alterations in important functions in the ovary like the balance between pro- and anti-apoptotic factors, follicular proliferation/apoptosis, and steroidogenesis, which may contribute to the follicular persistence and endocrine changes found in cattle with COD.

Ovarian localization of 11β-hydroxysteroid dehydrogenase (11βHSD): 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 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 11βHSD2 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 11βHSD 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 11βHSD1 in tertiary follicles and follicular cysts and a decrease in 11βHSD2 in follicular cysts. Moderate expression of 11βHSD1 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 11βHSD2 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 11βHSD1 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.

Alteration in the Expression Insulin-Like Growth Factor-II (IGF-II) Family Members in Bovine Cystic Ovarian Disease.

Alteration in the Expression Insulin-Like Growth Factor-II (IGF-II) Family Members in Bovine Cystic Ovarian Disease.

Veterinary clinical application of GnRH—questions of efficacy

The efficacy of GnRH treatments are reviewed in relation to prevention of embryo mortality, control of follicle development in synchronization programmes using PG as luteolysin, induction of ovulation in post-partum anoestrus and in bovine cystic ovarian disease. It is suggested that in cattle that GnRH is effective in increasing pregnancy rates when given either at the time of insemination (first or repeat) or between days 11 and 14 after insemination. Evidence is also presented for positive effects on pregnancy rates in sheep, mares and sows. Use of GnRH as an integral part of synchronising regimens where it is given 7 days before PG and then again 48–60 h after PG appears to be effective in increasing the synchrony of ovulation in controlled breeding programmes. The main synchronizing effect seems to reside in the second GnRH injection whereas the importance of the first is in prolonging the luteal phase in those cows treated late in the cycle. The published work on the potential use of GnRH to induce ovulation in anovulatory cattle is reviewed. Neither bolus dose injections, pulsatile, continuous infusion, nor controlled release formulations of GnRH, have yet proved effective in inducing fertile ovulations in a predictable or consistent manner. It is suggested that this is due to the variability of follicular status when treatment is initiated. GnRH is commonly used in the treatment of bovine cystic ovarian disease. However, although stimulating ovulation/luteinisation of a new follicle and luteinisation of the cyst, fertility of treated cattle remains very poor and it is suggested that a better understanding of the disease is needed before more effective treatments can be developed.

An ultrasonographic study of bovine cystic ovarian disease and its treatment

This study assessed the value of ultrasonography in characterising bovine cystic ovaries and monitoring their responses to different treatments. Thirteen cows were diagnosed by ultrasonography as having luteinised ovarian cysts and seven were diagnosed as having follicular ovarian cysts. Six of the former were treated with prostaglandin, four with a progesterone intravaginal device (PRID) and three with gonadotrophin-releasing hormone (Gn-RH); five of the latter were treated with Gn-RH and two with a PRID. All the animals were re-examined by ultrasound and blood was collected for the measurement of plasma progesterone concentration at intervals until oestrus. The treatment of the luteinised cysts with prostaglandin caused marked decreases in size and plasma progesterone concentration and altered their echotexture within two to four days; oestrus occurred within three to four days. In two of the cows treated with a PRID the cysts regressed within one to two weeks but the other two cows required supplementary treatment with prostaglandin; oestrus and ovulation were observed only after the cysts collapsed. Gn-RH stimulated oestrus and ovulation within three to four days but the cysts did not collapse until much later. The treatment of the follicular cysts with Gn-RH or a PRID caused fresh ovulation and the formation of a corpus luteum but had little immediate effect upon the cyst. The plasma progesterone concentrations in some of the cows with either follicular or luteal cysts were similar on the day of treatment and were therefore of little value in differentiating the types of cyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine cystic ovarian disease: plasma hormone concentrations and treatment.

Ovarian function in 91 dairy cows with cystic ovarian disease was assessed by rectal palpation and by plasma hormone analysis before and after treatment. Plasma analysis showed that 84% of the cysts were correctly classified clinically and only these cows are considered further. Luteinised cysts occurred in 59 cows whereas only 18 had non-luteinised cysts. The mean plasma concentrations of luteinising hormone (LH), follicular stimulating hormone (FSH), progesterone, oestradiol and testosterone were not significantly different when compared with values at relevant stages of the oestrous cycle in normal cows. Success of treatment with progesterone, a synthetic prostaglandin, human gonadotrophin (HCG), or gonadotrophin releasing hormone (GnRH) was not dependent upon prior hormone concentrations, except for the prostaglandin which required active luteal tissue. LH and FSH concentrations in cows with luteinised cysts were not significantly different before and after successful treatment with GnRH or progesterone. Normal luteal function was not always established after treatment of non-luteinised cysts with GnRH.