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-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.

Expression of TGFBR1, TGFBR2, TGFBR3, ACVR1B and ACVR2B is altered in ovaries of cows with cystic ovarian disease.

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.

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.

Effect of Parenteral Supplementation of Minerals and Vitamins on Oxidative Stress Biomarkers and Hepatic Fatty Acid Metabolism in Dairy Cows During the Transition Period.

In the present work we aimed to study the effects of parenteral vitamin and mineral supplementation on hepatic fatty acid metabolism as well as on the oxidative stress biomarkers in biological samples of transition cows. The supplemented group (SG, n = 11) received a subcutaneous injection of 5 mL of vitamin A palmitate 35 mg/mL, vitamin E acetate 50 mg/mL plus other injection of 5 mL of copper edetate 10 mg/mL, zinc edetate 40 mg/mL, manganese edetate 10 mg/mL, and sodium selenite 5 mg/mL on days - 60, - 30, and 7 (± 3) relative to calving. The control group (CG, n = 11) received two subcutaneous injections of 5 mL of 9 mg/mL sodium chloride at the same times of the SG. Blood, urine, and liver biopsies were sampled 21 (± 3) days before the expected calving date and 7 and 21 (± 3) days after calving. Results revealed that supplemented animals had higher glutation peroxidase (GSH-Px) activity, lower and higher concentration of 3-nitrotyrosine (3-NT) in the liver and plasma, respectively, higher expression of the mitochondrial beta-oxidation enzyme carnitine palmitoyltransferase 1 in the liver, and lower content of hepatic triacylglycerol, mirroring plasma liver function parameters. No differences between groups were found in the superoxide dismutase activity, MDA concentrations, the protein abundance of peroxisomal acyl-CoA oxidase 1, diacylglycerol O-acyltransferase 1, and peroxisome proliferator-activated receptor alpha. These results suggest that the vitamin and mineral supplementation provided to dairy cows had a beneficial effect on GSH-Px activity, hepatic 3-NT concentration, and on the metabolic adaptation during the peripartum period.

Biomarkers of oxidative stress and liver function in early lactation and their relationship with the reproductive efficiency of multiparous grazing dairy cows in Argentina. A retrospective study.

This study aimed to analyze the possible relationship between days to conception and different oxidative stress (OS) biomarkers and liver functional parameters in multiparous dairy cows. Besides, a fast reliable method for the accurate measurement of malondialdehyde (MDA) by liquid chromatography-tandem mass spectrometry was developed in several matrices. During lactation, the days to conception of 28 cows were determined for a retrospective study. According to this parameter, cows were divided into two groups: high and low days to conception (HDC and LDC, respectively). Blood, urine and liver biopsies were sampled 21 days before the expected calving date, and 7 and 21 days after calving. The method developed for MDA was validated according to international requirements. The lower limit of quantification was 0.25 µmol/L for plasma and urine and 10.00 µmol/L for liver tissue. No differences between groups were observed in the systemic concentration of non-esterified fatty acids, ß-hydroxybutyric acid and liver triacylglycerol content (P > 0.05). Cholesterol concentration was higher in the LDC than in the HDC group (P < 0.05). Plasma 3-nitrotyrosine (3-NT) concentration was lower in the LDC than in the HDC group on day 21 post-calving (P < 0.05). Superoxide dismutase activity was higher in the LDC than in the HDC group (P < 0.05). Particularly, in the liver, 3-NT and MDA concentrations were lower in the LDC than in the HDC group (P < 0.05). These results allow inferring that the amelioration of OS biomarkers in plasma and liver could be related to a better reproductive performance of dairy cows.

Alteration in localization of steroid hormone receptors and coregulatory proteins in follicles from cows with induced ovarian follicular cysts.

Cystic ovarian disease (COD) is an important cause of infertility in cattle. The altered follicular dynamics and cellular differentiation observed in COD may be mediated through a disruption of the expression of steroid receptors and their associated transcriptional cofactors. The aim of this study was to determine the protein expression profiles of ESR1, ESR2, PGR, AR, NCOA3, NCOR2, and PHB2 (REA) in ovarian follicles in an experimental model of COD induced by the administration of ACTH. Ovaries were collected and follicles were dissected from heifers during the follicular phase (control) or from heifers treated with ACTH to induce the formation of ovarian follicular cysts. Ovaries were fixed, sectioned, and stained immunohistochemically for steroid receptors and the associated transcription factors. The relative expression of ESR1 was similar in follicular cysts and in tertiary follicles from both control and cystic cows and was significantly higher than in secondary follicles. The expression of ESR2 in the granulosa was higher in cystic follicles. No differences were seen for PGR. The expression of androgen receptor was significantly increased in tertiary follicles with lower immunostaining in cysts. The expression of NCOA3 was observed in the granulosa and theca with a significantly increased expression in the theca interna of cystic follicles. The highest levels of NCOR2 expression in granulosa, theca interna, and theca externa were observed in cysts. In granulosa cells, NCOR2 levels increase progressively as follicles mature and the treatment had no effect. In summary, ovaries from animals with induced COD exhibited altered steroid receptor expression compared with normal animals, as well as changes in the expression of their regulators. It is reasonable to suggest that in conditions characterized by altered ovulation and follicular persistence, such as COD, changes in the intra-ovarian expression of these proteins could play a role in their pathogenesis.

Preclinical Efficacy and Toxicology Evaluation of RAC1 Inhibitor 1A-116 in Human Glioblastoma Models.

Malignant gliomas are the most common primary central nervous system tumor in adults. Despite current therapeutics, these tumors are associated with poor prognosis and a median survival of 16 to 19 months. This highlights the need for innovative treatments for this incurable disease. Rac1 has long been associated with tumor progression and plays a key role in glioma's infiltrative and invasive nature. The aim of this study is to evaluate the 1A-116 molecule, a Rac1 inhibitor, as targeted therapy for this aggressive disease. We found that targeting Rac1 inhibits cell proliferation and cell cycle progression using different in vitro human glioblastoma models. Additionally, we evaluated 1A-116 in vivo, showing a favorable toxicological profile. Using in silico tools, 1A-116 is also predicted to penetrate the blood-brain barrier and present a favorable metabolic fate. In line with these results, 1A-116 i.p daily treatment resulted in a dose-dependent antitumor effect in an orthotopic IDH-wt glioma model. Altogether, our study provides a strong potential for clinical translation of 1A-116 as a signal transduction-based precision therapy for glioma and also increases the evidence of Rac1 as a key molecular target.

Developmental Programming: Prenatal Testosterone Excess on Ovarian SF1/DAX1/FOXO3.

Prenatal testosterone (T) excess, partly via androgenic programming, enhances follicular recruitment/persistence in sheep as in women with polycystic ovarian syndrome (PCOS). Decreased anti-Mullerian hormone (AMH) in early growing and increased AMH in antral follicles may underlie enhanced recruitment and persistence, respectively. Changes in AMH may be mediated by steroidogenic factor 1 (SF1), an enhancer of AMH, and dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1), that antagonizes SF1. Another mediator could be forkhead box 03 (FOXO3) which regulates follicular recruitment/atresia. To test if androgen-programmed changes in SF1, DAX1, and FOXO3 proteins contribute to follicular defects in prenatal T-treated sheep, ovaries from control, prenatal T-, and dihydrotestosterone (DHT)-treated (days 30-90 of gestation) animals at fetal day (FD) 90, FD140, and 1 and 2 years-of-age were studied. Prenatal T increased DAX1 in granulosa cells of primordial through large preantral and theca cells of large preantral follicles at FD140 and increased SF1 in the granulosa cells of preantral and antral and theca cells of large preantral follicle at 2 years-of-age. Prenatal T increased FOXO3 only in theca cells of preantral (FD140) and antral (2 years-of-age) follicles. Prenatal DHT increased DAX1 in granulosa cells from small preantral follicles at FD140 while increasing SF1 in granulosa cells from antral follicles at 1 year-of-age. These age-dependent changes in DAX1/SF1 partly via androgen-programming are consistent with changes in AMH and may contribute to the enhanced follicular recruitment/persistence, and multifollicular phenotype of prenatal T-treated females and may be of translational relevance to PCOS.

Liver fatty acid metabolism associations with reproductive performance of dairy cattle.

The peri-calving period is characterized by a negative energy balance, which leads to lipid mobilization. Thus, during this period, the liver has important functions related to optimizing milk yield, preventing metabolic and infectious diseases, and improving fertility. To clarify the relationship between liver fatty acid metabolism and reproductive performance, the present study was conducted to assess the abundance of specific hepatic proteins related to lipid metabolism in both plasma and follicular fluid in dairy cattle with different days to conception (DC). Sixteen animals were grouped according to DC, as more and fewer DC (MDC and FDC, respectively). Blood and liver biopsies were sampled 14 days before the expected calving date and 4, 14 and 28 days after calving. The plasma beta-hydroxybutyric acid (BHBA) concentrations and the liver triacylglycerol (TAG) content were greater in the MDC group (P <  0.05), whereas the protein abundance of carnitine palmitoyl transferase 1 was greater in the FDC group (P < 0.05). Additionally, total bilirubin (TBil) concentration was less in the FDC than MDC group on day 28 (P < 0.05). These results indicate lipid mobilization and liver fatty acid oxidation capacity in dairy cows could contribute to the adaptations and reproductive performance.

Production and validation of a polyclonal serum against bovine FSH receptor.

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.

Characterization of persistent follicles induced by prolonged treatment with progesterone in dairy cows: an experimental model for the study of ovarian follicular cysts.

Cystic ovarian disease (COD) is a major factor contributing to poor reproductive efficiency of lactating dairy cows. The objective of the present study was to analyze the endocrine profile, growth dynamics, and histologic characteristics of persistent ovarian follicles-cysts developing in response to long-term administration of intermediate levels of progesterone. To this end, after synchronization of cows, a low dose of progesterone was administered for 5, 10, and 15 days after the expected day of ovulation in treated cows (groups P5, P10, and P15, respectively), using an intravaginal progesterone-releasing device. A significant increase in diameter was detected on Day 11 of progesterone treatment and thereafter (P < 0.05), and at Day 15 of persistence, the diameter of the persistent follicle reached a mean of 23 ± 0.6 mm. Microscopically, the persistent follicles had a complete granulosa, an intensely vascularized theca interna, and a collagenous theca externa layer. Temporal changes in the serum concentrations of estradiol, progesterone, and FSH were detected (effects of time, P < 0.01). Progesterone treatment completely inhibited the LH preovulatory surge in treated cows and affected the basal concentration of LH. The pulse frequency remained high at 5 and 10 days of persistence and declined (P < 0.05) after 15 days of persistence. The LH pulse concentration and pulse amplitude had a significant reduction (P < 0.05) during follicular persistence. Changes in the serum levels of estradiol, progesterone, 17-hydroxyprogesterone, and testosterone in serum and follicular fluid were also observed. In serum, estradiol increased gradually from proestrus to Day 10 of follicular persistence (P < 0.05), progesterone showed an increase (P < 0.05) at Day 5 of follicular persistence, 17-hydroxyprogesterone showed a significant decrease at 5 days of follicular persistence in relation to proestrus, and testosterone showed a significant increase (P < 0.05) from proestrus and Day 5 of persistence through Day 15 of follicular persistence. Correlation between serum and follicular fluid steroid concentrations was significant for testosterone (P < 0.0001) and not significant for estradiol and progesterone. These findings indicate that ovarian cysts in COD are similar in many ways to the persistent follicles induced by progesterone, with an analogous hormonal and morphologic context, thus confirming a local role of subluteal levels of progesterone in COD pathogenesis and in the regulatory mechanisms of the ovarian function.

Developmental programming: prenatal steroid excess disrupts key members of intraovarian steroidogenic pathway in sheep.

Prenatal testosterone (T) excess disrupts ovarian cyclicity and increases circulating estradiol levels as well as follicular recruitment and persistence culminating in multifollicular ovary similar to women with polycystic ovary syndrome. We tested whether prenatal T excess, by androgenic or estrogenic action, disrupts the steroid biosynthetic machinery in sheep in a cell-, follicle stage-, age-, and treatment-specific manner consistent with the ovarian disruptions and increased estradiol release. Impact of T/dihydrotestosterone (DHT) treatments from days 30-90 of gestation on steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase, cytochrome P-450 17α-hydroxylase/C17, 20-lyase (CYP17A1), and cytochrome P-450 aromatase (CYP19A1) were examined on fetal day 90, 140 and 10 months (postpubertal), and 21 months (adult, no DHT group) of age by immunohistochemistry. All 4 markers changed in a cell-, follicle stage-, and age-specific manner. Both treatments increased steroidogenic acute regulatory protein expression in preantral follicles of postpubertal and adult females. Effects of prenatal T and DHT on 3ß-hydroxysteroid dehydrogenase differed in a follicle- and age-specific manner. CYP17A1 was reduced in the theca interna of antral follicles by T, but not DHT, in 10- and 21-month-old females. CYP19A1 was reduced by both T and DHT at all ages barring an increase on fetal day 140. Reduced granulosa CYP19A1 and thecal CYP17A1 in adults likely disrupt the intrafollicular androgen/estrogen balance contributing to follicular persistence. The reduced thecal CYP17A1 expression suggests that the hyperandrogenic ovarian phenotype may originate from increased enzyme activity or alternatively via a different isoform of CYP17. The reduced CYP19A1 in antral follicles of adults indicates that the increased circulating estradiol release likely arises from the increased number of persisting follicles.

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 ßA-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 ßB) levels in serum and follicular fluid. We found an increased expression of the ßA-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 ßB) 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.

Changes in the expression of Heat Shock Proteins in ovaries from bovines with cystic ovarian disease induced by ACTH.

Cystic ovarian disease (COD), which is considered one of the most important causes of reproductive failure in dairy cattle, induces intraovarian changes in the expression of numerous genes. The purpose of this study was to analyze the changes in the expression of Heat Shock Proteins (HSPs) in ovaries from bovines with cystic ovarian disease induced by ACTH. Immunoreactivity for Heat Shock Proteins (HSPs) in ovaries of cows with induced COD showed differential expression patterns in growing follicles from the control group. The immunopositive area for Hsp27 and Hsp60 in granulosa cells showed significant differences between tertiary follicles from normal cycling animals and those from animals with induced COD. The cysts showed increased Hsp27 immunostaining in theca cells in relation to tertiary follicles from normal cycling cows. Hsp70 immunostaining was more intense in cystic follicles than in other follicular categories from animals with induced COD, in both granulosa and theca cells. In granulosa cells, tertiary follicles from the control group showed higher levels of Hsp90 than cysts. These results demonstrate that there are differences in HSP protein expression when COD is induced. In fact, HSP expression would be part of the functional response to the changes in hormones and neurotransmitters induced by stress, indicating that HSPs can control hormonal functions and vice versa.