G.J. Hein

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.

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.

Follicular Cysts: A Single Sign and Different Diseases. A View from Comparative Medicine

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.

Liquid chromatography tandem-mass spectrometry (LC-MS/MS) and dried blood spot sampling applied to pharmacokinetics studies in animals: Correlation of classic and block design:

A relative bioavailability study (RBA) of two phenytoin (PHT) formulations was conducted in rabbits, in order to compare the results obtained from different matrices (plasma and blood from dried blood spot (DBS) sampling) and different experimental designs (classic and block). The method was developed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in plasma and blood samples. The different sample preparation techniques, plasma protein precipitation and DBS, were validated according to international requirements. The analytical method was validated with ranges 0.20–50.80 and 0.12–20.32 µg ml−1, r > 0.999 for plasma and blood, respectively. Accuracy and precision were within acceptance criteria for bioanalytical assay validation (< 15 for bias and CV% and < 20 for limit of quantification (LOQ)). PHT showed long-term stability, both for plasma and blood, and under refrigerated and room temperature conditions. Haematocrit values were measured during the validation process and RBA study. Finally, the pharmacokinetic parameters (Cmax, Tmax and AUC0–t) obtained from the RBA study were tested. Results were highly comparable for matrices and experimental designs. A matrix correlation higher than 0.975 and a ratio of (PHT blood) = 1.158 (PHT plasma) were obtained. The results obtained herein show that the use of classic experimental design and DBS sampling for animal pharmacokinetic studies should be encouraged as they could help to prevent the use of a large number of animals and also animal euthanasia. Finally, the combination of DBS sampling with LC-MS/MS technology showed to be an excellent tool not only for therapeutic drug monitoring but also for RBA studies.

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.

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.

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.