Effects of adrenocorticotrophic hormone on the expression of matrix metalloproteinases and their inhibitors in the bovine ovary.

Cattle undergo numerous environmental and management stressors that reduce fertility and affect ovulation. The extracellular matrix of the follicle wall can be altered by matrix metalloproteinases (MMPs), the activities of which are regulated by interleukins and tissue-specific inhibitors of metalloproteinases (TIMPs), especially during ovulation. The aims of the present study were to: (1) evaluate changes in the hormone milieu, the localisation and activity of MMP2 and MMP9 and the localisation of MMP14, TIMP1 and TIMP2 in response to adrenocorticotrophic hormone (ACTH) during the preovulatory period in cows; and (2) determine the direct effects of ACTH on the mRNA expression of MMP2 and MMP9 in the cultured follicle wall of bovine ovaries obtained from an abattoir. 100IU ACTH was administered during pro-oestrus every 12h until ovariectomy, which was performed before ovulation. Cortisol concentrations in the plasma and follicular fluid (FF) of preovulatory follicles were higher in ACTH-treated than control cows. Progesterone presented subluteal concentrations in plasma of ACTH-treated cows (P<0.05). MMP2 immunostaining and activity in ovaries were higher in ACTH-treated than control cows (P<0.05), whereas MMP9 immunostaining was similar between the two groups. However, unlike in control cows, MMP9 activity was absent in the FF of ACTH-treated cows. These results suggest that the administration of ACTH during the preovulatory period in cows could cause changes that culminate in modifications in the content and activation of MMPs and TIMPs in the ovary, which could interfere with the ovulation process.

Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

Detection and activity of 11 beta hydroxylase (CYP11B1) in the bovine ovary.

Glucocorticoids (GCs) such as cortisol and corticosterone are important steroid hormones with different functions in intermediate metabolism, development, cell differentiation, immune response and reproduction. In response to physiological and immunological stress, adrenocorticotropic hormone (ACTH) acts on the adrenal gland by stimulating the synthesis and secretion of GCs. However, there is increasing evidence that GCs may also be synthesized by extra-adrenal tissues. Here, we examined the gene and protein expression of the enzyme 11ß-hydroxylase P450c11 (CYP11B1), involved in the conversion of 11-deoxycortisol to cortisol, in the different components of the bovine ovary and determined the functionality of CYP11B1 in vitro CYP11B1 mRNA was expressed in granulosa and theca cells in small, medium and large antral ovarian follicles, and CYP11B1 protein was expressed in medium and large antral follicles. After stimulation by ACTH, we observed an increased secretion of cortisol by the wall of large antral follicles. We also observed a concentration-dependent decrease in the concentration of cortisol in response to metyrapone, an inhibitor of CYP11B1. This decrease was significant at 10-5 µM metyrapone. In conclusion, this study demonstrated for the first time the presence of CYP11B1 in the bovine ovary. This confirms that there could be a local synthesis of GCs in the bovine ovary and therefore a potential endocrine responder to stress through these hormones.

Immunohistochemical expression of growth factors in the follicular wall of normal and cystic ovaries of sows.

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF-I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF-I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF-II was very similar to pattern noted in IGF-I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF-II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.

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.

Cellular proliferation rate and insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 and estradiol receptor alpha expression in the mammary gland of dairy heifers naturally infected with gastrointestinal nematodes during development.

Mammary ductal morphogenesis during prepuberty occurs mainly in response to insulin-like growth factor-1 (IGF-1) and estradiol stimulation. Dairy heifers infected with gastrointestinal nematodes have reduced IGF-1 levels, accompanied by reduced growth rate, delayed puberty onset, and lower parenchyma-stroma relationship in their mammary glands. Immunohistochemical studies were undertaken to determine variations in cell division rate, IGF-1 system components, and estradiol receptors (ESR) during peripubertal development in the mammary glands of antiparasitic-treated and untreated Holstein heifers naturally infected with gastrointestinal nematodes. Mammary biopsies were taken at 20, 30, 40, and 70 wk of age. Proliferating cell nuclear antigen immunolabeling, evident in nuclei, tended to be higher in the parenchyma of the glands from treated heifers than in those from untreated. Insulin-like growth factor binding proteins (IGFBP) type 2 and type 3 immunolabeling was cytoplasmic and was evident in stroma and parenchyma. The IGFBP2-labeled area was lower in treated than in untreated heifers. In the treated group, a maximal expression of this protein was seen at 40 wk of age, whereas in the untreated group the labeling remained constant. No differences were observed for IGFBP3 between treatment groups or during development. Immunolabeling for α ESR (ESR1) was evident in parenchymal nuclei and was higher in treated than in untreated heifers. In the treated group, ESR1 peaked at 30 wk of age and then decreased. These results demonstrate that the parasite burden in young heifers negatively influence mammary gland development, affecting cell division rate and parameters related to estradiol and IGF-1 signaling in the gland.

Epidemiological description of cystic ovarian disease in argentine dairy herds: risk factors and effects on the reproductive performance of lactating cows.

To describe the epidemiology of cystic ovarian disease (COD), to find possible risk factors associated with the incidence of cysts and to analyse the impact of COD on the reproductive performance of dairy cows, databases from 22 dairy herds from the main dairy region in Argentina were retrospectively evaluated throughout a 3-year period (2009-2011). A total of 248 COD cases over 9156 parturitions were recorded, resulting in a cumulative incidence rate of 2.7%. Cystic ovarian disease incidence density was lower during the first 100 days post-partum (DPP) than during later stages of lactation. Seasonality had a significant influence on the disease presentation with higher incidence rates during winter and spring. Cows with a previous diagnosis of clinical mastitis showed 2.72 times more chances of developing ovarian cysts. Cystic cows had longer calving to first service and calving to conception intervals and lower conception rate than controls.

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.

Intermediate filament proteins expression and carbohydrate moieties in trophoblast and decidual cells of mature cat placenta.

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Evaluation of the expression of growth hormone and its receptor during the resumption of postpartum ovarian follicle development in dairy cows.

Growth hormone is a key endocrine factor for metabolic adaptations to lactation and optimal reproductive function of the dairy cow. This study aimed to analyze the expression of GH and its receptor (GHR) in ovarian follicles, along with metabolic biomarkers, during the resumption of the postpartum follicular development, and to analyze the immunolocalization and protein expression of GH and GHR in preovulatory follicles. Thirty-six dairy cows were grouped according to the postpartum days (PPD) until the establishment of the first dominant follicle in: cows that established their first dominant follicle at fewer postpartum days (FPPD group; n = 15) and cows that established their first dominant follicle at more postpartum days (MPPD group; n = 22). For a second analysis, the same cows were regrouped according to the calving season (S), into cows calving in autumn (n = 20) and cows calving in winter (n = 17). During the PP, blood and follicular aspirates were obtained at two timepoints (T): when the first dominant follicle was established (T1, day 9 ± 2), and when the preovulatory follicle was established (T2, day 45 ± 2). Also, six dairy cows were ovariectomized in proestrus and ovarian histological sections were obtained. Growth hormone mRNA was detected in granulose cells from ovarian follicle sampled during PP. A PPD × T interaction was observed for GHR mRNA, where it was greater in the FPPD cows than in the MPPD cows at T1. Metabolic biomarkers and reproductive hormones showed differences or interaction between PPD, T, S, depending on the case. Also, GH and GHR were immunolocalized in granulosa and theca interna cells of preovulatory follicles. These results confirm the expression of GH and GHR in the mature ovarian follicles of dairy cows and show a possible association between greater GHR expression and an earlier resumption of postpartum follicular development.

Lectin-binding pattern in ovarian structures of rats with experimental polycystic ovaries.

Numerous experimental models in different species have been developed for the study of polycystic ovarian syndrome. In this study, we used a model of induction of polycystic ovaries (PO) in rats by exposure to constant light to study the distribution and variations of glycosylated residues present in the different ovarian structures. Seven biotinylated lectins were used (Con-A, WGA, DBA, SBA, PNA, RCA and UEA-I) on tissue sections, and detection was performed using the streptavidin/peroxidase method. In tissue sections was observed an increase in affinity for Con-A in the granulosa and theca interna of growing follicles and cysts in animals with PO in relation to the control group. Follicular cysts showed higher affinity for WGA and RCA-I which growing follicles in the same group, and there was a decrease in affinity for PNA in the cysts in relation to the growth of follicles in both groups. Atretic follicles in both groups showed greater labelling with lectins PNA, SBA and RCA-I in relation to healthy follicles. It could also be noted that the zona pellucida of cystic follicles lost the affinity for the lectin Con-A. There was no staining on follicles in any category with the lectins DBA and UEA-I, although it was staining in the corpus luteum (control group) and in the mesothelium and interstitial glands of both groups with DBA. These observations probably reflect changes in the glycosaminoglycans present in the different ovarian compartments or in the glycosylation of cellular components essential for proper follicular dynamics.

Apoptosis-related protein expression during pre- and post-natal testicular development after administration of glucocorticoid in utero in the sheep.

Pre-natal glucocorticoids are used in women at risk of preterm delivery to induce foetal lung maturation. However, glucocorticoids can produce negative outcomes for other tissues such as the reproductive system. We therefore tested the effects of pre-natal betamethasone on testicular morphology and apoptotic protein immune expression during pre- and post-natal development. Pregnant ewes (n = 42) bearing singleton male foetuses were randomly allocated to receive intramuscular injections of saline or betamethasone (0. 5 mg/kg) at 104, 111 and 118 days of gestation (DG). Testes were collected at 121 and 132 DG, and at 45 and 90 post-natal days (PD) and subjected to morphometric analysis (volume densities of sex cords and interstitial tissues; sex cord diameter). Immunohistochemistry (% stained area) was used to assess active caspase-3, Bax, Bcl-2 and cell-cycle proteins (PCNA). Compared with control values, betamethasone treatment decreased sex cord diameter at 121 DG, 45 and 90 PD, and sex cord volume at 90 PD. Active caspase-3 was decreased by betamethasone at 121 DG and 90 PD, but Bax was increased in all betamethasone groups. Bcl-2 and PCNA decreased in the betamethasone groups at 121 DG and 45 PD, but increased at 132 DG and 90 PD. We conclude that high levels of pre-natally administered glucocorticoid reduce foetal testicular development, perhaps via changes in the balance between pro- and anti-apoptotic proteins and cell-cycle proteins. These outcomes could compromise the future spermatogenic potential of male offspring.

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.

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.

Exogenous ACTH stimulus during the preovulatory period alters patterns of leukocyte recruitment in the ovary of dairy cows.

Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C-C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary.

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.

Association of glucocorticoid receptor expression with key members of the insulin signaling pathway and heat shock proteins in the bovine ovary.

Glucocorticoids (GCs) act through their receptor (GR) as regulators in different biological processes such as reproduction. In the absence of GCs, the GR remains inactive in the cytoplasm by associating with heat shock proteins (HSPs), which act as molecular chaperones, among which the most relevant are HSP90 and HSP70. Cytoplasmic GC-activated GR mediates non-genomic effects, interacting with members of signaling pathways such as PI3K/Akt, which participates in several metabolic processes, including the insulin signaling pathway. The aim of the present study was to evaluate possible associations between the cytoplasmic GR and the main intermediates of the insulin signaling pathway and HSP90 and HSP70 in ovaries of dairy cows. To this end, the protein expression of cytoplasmic GR, key members of the insulin signaling pathway, and HSPs was evaluated in ovarian preovulatory follicles of non-lactating Holstein cows in proestrus. Positive associations were observed between protein expression of GR and HSP90, IRS1, pIRS1, PI3K and pAkt (p < 0.05; ß > 0) in granulosa cells of dominant follicles of dairy cows. Instead, in theca cells, no associations were observed between protein expression of GR and members of the insulin signaling pathway or HSPs. These data provide evidence of the possible association between the non-genomic mechanisms of action of the GR and the insulin signaling pathway in the bovine ovary.

Hormonal regulation of female reproduction.

Reproduction is an event that requires the coordination of peripheral organs with the nervous system to ensure that the internal and external environments are optimal for successful procreation of the species. This is accomplished by the hypothalamic-pituitary-gonadal axis that coordinates reproductive behavior with ovulation. The primary signal from the central nervous system is gonadotropin-releasing hormone (GnRH), which modulates the activity of anterior pituitary gonadotropes regulating follicle stimulating hormone (FSH) and luteinizing hormone (LH) release. As ovarian follicles develop they release estradiol, which negatively regulates further release of GnRH and FSH. As estradiol concentrations peak they trigger the surge release of GnRH, which leads to LH release inducing ovulation. Release of GnRH within the central nervous system helps modulate reproductive behaviors providing a node at which control of reproduction is regulated. To address these issues, this review focuses on several critical questions. How is the HPG axis regulated in species with different reproductive strategies? What internal and external conditions modulate the synthesis and release of GnRH? How does GnRH modulate reproductive behavior within the hypothalamus? How does disease shift the activity of the HPG axis?

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.

MC2R/MRAP2 activation could affect bovine ovarian steroidogenesis potential after ACTH treatment.

Stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, reducing fertility by interfering with the mechanisms that regulate the timing of events within the follicular phase of the estrous cycle. In the HPA axis, melanocortin 2 receptor (MC2R) mediates responses to adrenocorticotropic hormone (ACTH) in concert with melanocortin receptor accessory protein 2 (MRAP2). The aims of the present study were: (1) to evaluate the effects of ACTH administered in cows in the preovulatory period on the expression of the MC2R/MRAP2 complex in the dominant follicle; and (2) to analyze the involvement of Extracellular signal Regulated Kinase 1 (ERK1) signaling in the activation of MC2R and the expression of key enzymes involved in the biosynthesis of glucocorticoids (GCs) in the dominant follicle. To this end, 100 IU ACTH was administered to Holstein cows from a local dairy farm during pro-estrus every 12 h for four days until ovariectomy, which was performed before ovulation. Protein immunostaining of MC2R was higher in the dominant follicles of ACTH-treated cows (p < 0.05). Also, Western blot analysis showed higher activation of the ERK1 signaling pathway in ACTH-treated cows (p < 0.05). Finally, immunohistochemistry performed in the dominant follicles of ACTH-treated cows detected higher expression of CYP17A1 and CYP21A2 (p < 0.05). These results suggest that the bovine ovary is able to respond locally to ACTH as a consequence of stress altering the expression of relevant steroidogenic enzymes. The results also confirm that the complete GC biosynthesis pathway is present in bovine dominant follicle and therefore GCs could be produced locally.