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Context The establishment of pregnancy in cows requires uterine activity regulation of the main Hippo signalling effector yes-associated protein 1 (YAP). It remains unknown (1) how YAP activity at the corpus luteum (CL) correlates with early pregnancy-related events in ruminants; and (2) if YAP activity in the uterus and CL can be affected by metabolic disorders that may lead to pregnancy failure in ruminants. Aims and methods To determine the effect of early pregnancy on total and phospho-YAP expression and its transcriptional activity in the CL, we compared non-pregnant vs pregnant ewes. To understand the YAP activity dysregulation with disorders that may result in pregnancy loss, we induced negative energy balance in pregnant ewes. Key results and conclusions Our main results indicate that early pregnancy alters the expression and activity patterns of YAP in the ovine CL but not in the endometrium. In addition, while our NEB-induced model fails to alter YAP activity at the endometrium level, we found that fasting during the first but not second week of pregnancy affects YAP activity in the CL of pregnant ewes. Implications The data presented herein provide considerable insight into the activity of a signalling pathway that may be a key player in pregnancy recognition and establishment in ewes.

Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. β-hydroxybutyrate (BHBA) is the main circulating ketone body, which accumulates within follicular fluid. The aim of this study was to evaluate the effects of BHBA on follicle growth and on ovulatory mechanisms in cattle. At 72 h after intrafollicular injection, there was a decrease in follicular diameter in BHBA group compared to control (P = 0.02). Furthermore, follicle growth rate was reduced post-treatment with BHBA in comparison to the control group (P 0.03). The BHBA intrafollicular injection in follicles ≥ 12 mm, however, did not affect E2 and P4 concentrations in the follicular fluid. In addition, the relative abundance of genes involved in the ovulatory cascade (ADAM 17, AREG, EREG, PTGS2), steroidogenesis (CYP19A1, 3BHSD, STAR), cellular stress (SOD1, CAT, GPX1, HSPA5, XBP1s, XBP1u, ATF4, ATF6), monocarboxylic acid transporters (SLC16A1, SLC16A7) and apoptosis (XIAP) was similar between groups. In conclusion, the results of this study indicate that the increase in intrafollicular concentrations of BHBA affects follicular growth, but it does not compromise the ovulatory cascade and cellular homeostasis in bovine granulosa cells.

The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 μM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.

Background Disruption of the balance between the production of ROS and their removal through enzymatic and non-enzymatic (antioxidant) processes has been proposed as a new mechanism in the pathology of polycystic ovary syndrome (PCOS). Evidence from animal models of PCOS (prenatally androgenized sheep) has suggested that treatment with insulin sensitizers, but not antiandrogens, can reduce increases in ROS. Materials and methods In the present study, we investigated the effects of neonatal treatment with a gonadotropin-releasing hormone (GnRH) agonist (leuprolide acetate) on prenatally androgenized sheep with testosterone propionate to determine its impact on oxidative stress molecules (ferric reducing antioxidant power [FRAP], advanced oxidation protein product [AOPP], nitric oxide [NOx], albumin) at 8, 12, and 18 months of age. Results Androgenized ewes (but not leuprolide-treated ewes) showed reduced total cholesterol levels associated with a decrease in the ratio of visceral to subcutaneous adiposity (adjusted to abdominal area) as determined by computed tomography. In androgenized ewes at 12 months of age, an increase in subcutaneous fat and relative decrease in the visceral fat compartment did not affect the expression of REDOX markers. At 18 months of age, however, the levels of NOx metabolites decreased in androgenized animals, but remained close to normal in ewes subjected to neonatal treatment with leuprolide acetate. Other oxidative stress parameters (FRAP, AOPP, albumin) did not vary among groups. Conclusion Our results demonstrate that the GnRH agonist leuprolide (as a single dose after birth) had weak effects on markers of the oxidative stress balance.

Failure to ovulate is a major cause of infertility. The critical pathway that induces ovulation involves the EGF and MAPK phosphorylation, but studies in rodents have suggested that the Hippo activator, YAP, is also involved. It is unknown whether YAP-dependent transcriptional activity is important for the LH- or EGF-induced ovulatory cascade in monovulatory species such as the cow. Using a well-defined preovulatory GC culture system, we employed pharmacological inhibitors to demonstrate that YAP signaling is critical for expression of EGFR and downstream target genes EREG, EGR1 and TNFAIP6. Most importantly, by using an ultrasound guided follicle injection system, we also showed that the classic Hippo signaling inhibitor Verteporfin inhibits GnRH-induced ovulation in vivo in cattle. In conclusion, YAP transcriptional activity is critical for EGF-like cascade induced by LH to promote ovulation in a monovulatory species.

There is growing evidence that greater than homeostatic blood concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyrate (BHBA) have negative consequences on dairy cow’s fertility, but effects on cell homeostasis in the reproductive system is not completely understood. In this study, lipids accumulation, reactive oxygen species (ROS) concentrations, abundance of gene transcripts, and immunofluorescence signal of H3K4me3 and H3K9me3 were evaluated in endometrial epithelial cells of cattle cultured with NEFAs (Oleic (OA), Stearic (SA) and Palmitic (PA) acids), BHBA, NEFAs + BHBA or each of the three NEFAs alone. The cellular lipids were in greater concentrations as a result of NEFAs + BHBA, NEFAs, SA or OA supplementation, but not by BHBA or PA. The ROS concentrations were greater when there were treatments with NEFAs + BHBA, NEFAs or BHBA. The relative mRNA abundance for genes involved in the regulation of apoptosis (XIAP), glucose transport (GLUT3), and DNA methylation (DNMT1) were greater when there were NEFAs + BHBA, but not NEFAs, BHBA, OA, SA or PA treatments. The immunofluorescence signal for H3K9me3 was greater when there were NEFAs + BHBA, NEFAs or PA, but not by BHBA, OA or SA treatments. These findings indicate that NEFAs and BHBA have an additive effect on endometrial cells of cattle by altering epigenetic markers and the expression of genes controlling important cellular pathways. Furthermore, there was cellular lipid accumulation and increased H3K9me3 in cultured bovine endometrial cells that was mainly induced by OA and PA treatments, respectively.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Dairy cows frequently undergo a state of negative energy balance (NEB) after parturition and some have impaired ovarian functions that result in delayed resumption of estrous cyclicity and development of follicles without ovulation occurring. During the postpartum period, cows undergo body-fat store losses, hormonal changes, fat mobilization and increases in nonesterified fatty acid (NEFAs) concentrations in blood and follicular fluid. The effect of NEFAs on follicular development and function of follicular cells, however, is not fully understood. The aim of this study, therefore, was to study the effect of an intrafollicular injection of a mixture of oleic, stearic and palmitic NEFAs on dominant follicle development and function of granulosa cells in cows that were not in a NEB state. Follicular size was less at 24 and 48 h after administration of NEFAs compared to that of control follicles injected with vehicle only. At 24 h after intrafollicular injection, the relative mRNA transcript abundance for proteins involved in steroidogenesis (CYP19A1, 3BHSD, STAR, FSHR), metabolism (GLUT1, GLUT3, INSR, IRS1, IRS2, SLC27A1, PPARG), and cell proliferation and apoptosis (CCND2; XIAP) in granulosa cells, as well as estradiol concentrations in follicular fluid were similar in control and NEFA-treated follicles. In conclusion, the results of this study indicate increased intrafollicular concentrations of NEFAs in cows that are not in a NEB state has a detrimental effect on follicle development. We propose intrafollicular injection is a useful approach to further investigate the local effects of NEFAs on the function of follicular cells.

The main clinical feature associated with hyperandrogenism in polycystic ovary syndrome (PCOS) in humans is hirsutism, where hair increases its length, pigmentation, and particularly its diameter. Currently, it is not known whether PCOS animal models also exhibit changes in the hair. Therefore, the aim of this study was to explore the wool characteristics in sheep prenatally androgenized (PA) with testosterone propionate. After 4 and 13 months of life, wool was collected from the top of the shoulder of both females and males (both androgenized and controls). The offspring sheep were followed for up to 19 months of life to evaluate testosterone and androstenedione serum levels by ultra-high-performance liquid chromatography-tandem mass spectrometry, determine insulin and glucose response to intravenous glucose tolerance test, and address estrus cyclicity during the second breeding season. PA male animals showed a reduction in wool fiber diameter at 4 months of age compared with controls (P = 0.02) but not at 13 months, whereas PA females showed increased hair diameter at 13 months (P = 0.002), with no difference at 4 months. No substantial changes in other hair parameters (length, color, and medullation) were identified. In addition, increased levels of serum testosterone were observed in PA female sheep compared with controls at 12 months (P = 0.03). Our results indicate for the first time, to our knowledge, that changes in wool fiber diameter observed in PA ewes replicate, at the translational level, the increase in hair diameter in hirsute women with PCOS.

During folliculogenesis, the luteinizing hormone (LH) surge triggers dynamic events in granulosa cells that culminate with ovulation. The aim of this study was to evaluate if the epidermal growth factor receptor (EGFR) is required for ovulation in cattle, and if it regulates the expression of the natriuretic peptide (NP) system in granulosa cells after gonadotropin-releasing hormone (GnRH)/LH stimulation. It was observed that GnRH induces amphiregulin (AREG) and epiregulin (EREG) mRNA at 3 and 6 h after in vivo treatment, but the expression of these genes was not regulated by atrial (ANP) and C-type (CNP) NPs in granulosa cells cultured in vitro. The abundance of mRNA encoding the NP receptors (NPR1, 2 and 3) was not altered by LH supplementation and/or EGFR inhibition (AG1478; AG) in granulosa cells after 6 h of in vitro culture. However, in the same conditions, mRNA encoding the natriuretic peptide precursor C (NPPC) was upregulated by LH, whereas AG (0.5 and 5 μM) inhibited the LH effect. In order to confirm those results, 5 μM AG or saline were intrafollicularly injected in preovulatory follicles and cows were simultaneously treated with GnRH intramuscularly. Granulosa cells harvested at 6 h after GnRH injection revealed higher NPR3 and lower NPPC mRNA levels in AG-treated, compared to control cows. However, intrafollicular injection of AG did not inhibit GnRH-induced ovulation. In granulosa cells cultured in vitro, ANP associated with LH increased prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA abundance. In conclusion, we inferred that LH modulated NPPC and NPR3 mRNA abundance through EGFR in bovine granulosa cells, but ovulation in cattle did not seem to depend on EGFR activation.

Lady Serrano Mujica,1 Alessandra Bridi,1,2 Ricardo Della Méa,1 Vitor Braga Rissi,1 Naiara Guarda,3 Rafael Noal Moresco,3 Melissa Orlandin Premaor,4 Alfredo Quites Antoniazzi,1 Paulo Bayard Dias Gonçalves,1 Fabio Vasconcellos Comim1,4 1Laboratory of Biotechnology and Animal Reproduction, BioRep, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, 2Department of Veterinary Medicine, University of São Paulo, São Paulo, 3Laboratory of Clinical Biochemistry, Department of Clinical and Toxicological Analysis, 4Department of Clinical Medicine, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, Brazil Background: Several studies have described an enhanced inflammatory status and oxidative stress balance disruption in women with polycystic ovary syndrome (PCOS). However, there is scarce information about redox markers in the blood of androgenized animal models. Here, we evaluated the serum/plasma oxidative stress marker and metabolic parameter characteristics of prenatal (PreN) and postnatal (PostN) androgenized rat models of PCOS. Materials and methods: For PreN androgenization (n=8), 2.5mg of testosterone propionate was subcutaneously administered to dams at embryonic days 16, 17, and 18, whereas PostN androgenization (n=7) was accomplished by subcutaneously injecting 1.25mg of testosterone propionate to animals at PostN day 5. A unique control group (n=8) was constituted for comparison. Results: Our results indicate that PostN group rats exhibited particular modifications in the oxidative stress marker, an increased plasma ferric-reducing ability of plasma, and an increased antioxidant capacity reflected by higher albumin serum levels. PostN animals also presented increased total cholesterol and triglyceride–glucose levels, suggesting severe metabolic disarrangement. Conclusion: Study findings indicate that changes in oxidative stress could be promoted by testosterone propionate exposure after birth, which is likely associated with anovulation and/or lipid disarrangement. Keywords: animal models of PCOS, oxidative stress, prenatal, postnatal

Prostaglandin F2α (PGF) induces the precipitous loss of steroidogenic capabilities and cellular death in the corpus luteum of many species, yet the molecular mechanisms underlying this event are not completely understood. Signal transducer and activator of transcription 3 (STAT3) was activated in granulosa cells during follicle atresia, whereas AKT is immediately down-regulated in the corpus luteum after PGF treatment in cattle; however, their involvement in both functional and morphological luteolysis in monovular species still need to be determined. Blood samples and corpus lutea were collected from cows before (0) and 2, 12, 24, and 48 hr after PGF treatment on Day 10 of the estrous cycle (4-5 cows per time point). Serum progesterone concentrations decreased by threefold (p < 0.05) within 2 hr, confirming functional luteolysis. The mRNA abundance of the pro-apoptotic gene BAX increased 12-48 hr post-PGF treatment (p < 0.05), while morphological luteolysis was observed 24 and 48 hr after PGF treatment, based on the loss of plasma membrane integrity, reduction of cytoplasmic volume, and pyknotic nuclei. Phosphorylated STAT3 increased, peaking at 12 hr, and remained elevated until 48 hr after PGF treatment. SOCS3 transcript abundance also increased (p < 0.05) starting at 2 hr post-PGF treatment. In contrast, AKT phosphorylation decreased by 12 hr after treatment. Thus, activation of STAT3 and inactivation of AKT signaling are involved in structural regression of the corpus luteum.

Obesity reaches an epidemic level worldwide, and this condition is associated with chronic low-grade inflammation and secondary comorbidities, largely driven by global changes in lifestyle and diet. Various dietary approaches are proposed for the obesity treatment and its associated metabolic disorders. Good taste, antioxidant functions, and vitamins have been attributed to virgin coconut oil (VCO). However, VCO contains a large amount of saturated fatty acids, and the consumption of this fat is associated with a number of secondary diseases. We evaluate the effects of VCO supplementation on biochemical, inflammatory, and oxidative stress parameters in rats fed with high-fat diet (HFD). After feeding with HFD for 12 weeks, the animals were supplemented with VCO for 30 days. HFD+VCO group increased in diet intake, weight gain, low-density lipoprotein cholesterol level, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These findings were accompanied by increased in hepatic lipid profile and fat deposition in the liver. Adipocyte hypertrophy was observed in the HFD+VCO group, which was associated with elevated expression of tumor necrosis factor alpha (TNF-α) in adipose tissue. These results revealed that VCO associated with HFD induced important metabolic alterations, adipose inflammation, and hepatic lipid accumulation in rats.

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control, locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functions in vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17β-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control −ve). The addition of 5.0-mM L-arg increased (P 0.05) at 22 hours. All concentrations of L-arg tested increased (P 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control −ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control −ve and treatment with 5-mM L-arg progressively decreased (P

Long-term administration of valproic acid (VPA) is known to promote reproductive impairment mediated by increase in testicular oxidative stress. Vitamin E (VitE) is a lipophilic antioxidant known to be essential for mammalian spermatogenesis. However, the capacity of this vitamin to abrogate the VPA-mediated oxidative stress has not yet been assessed. In the current study, we evaluated the protective effect of VitE on functional abnormalities related to VPA-induced oxidative stress in the male reproductive system. VPA (400 mg kg −1 ) was administered by gavage and VitE (50 mg kg −1 ) intraperitoneally to male Wistar rats for 28 days. Analysis of spermatozoa from the cauda epididymides was performed. The testes and epididymides were collected for measurement of oxidative stress biomarkers. Treatment with VPA induced a decrease in sperm motility accompanied by an increase in oxidative damage to lipids and proteins, depletion of reduced glutathione and a decrease in total reactive antioxidant potential on testes and epididymides. Co-administration of VitE restored the antioxidant potential and prevented oxidative damage on testes and epididymides, restoring sperm motility. Thus, VitE protects the reproductive system from the VPA-induced damage, suggesting that it may be a useful compound to minimize the reproductive impairment in patients requiring long-term treatment with VPA.

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400 mg kg −1 ) by gavage for 28 days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10 mg kg −1 ) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment.

We analysed the effects of quercetin-containing diet on blood parameters, antioxidant status and pituitary hormone expression in silver catfish. Diets containing three concentrations of quercetin (0, 0.15 and 0.30%) were provided to fish once a day. The results indicated that quercetin did not promote any significant change on the haematological and biochemical parameters measured. Fish that received the diet with quercetin presented decreased lipid peroxidation (LPO) (measured by lipid hydroperoxides and thiobarbituric acid reactive substances) in all tissues evaluated. On the other hand, the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were higher in tissues of fish fed with diets containing quercetin. Additionally, the content of non-protein thiols, total reactive antioxidant potential and ascorbic acid were also higher in tissues of quercetin fed fish. Finally, there was no changes regarding cortisol levels and the expression of growth hormone, prolactin and somatolactin in fish fed with quercetin when compared with the control. Our results suggests that supplementation of silver catfish diet with quercetin is beneficial since it reduced the LPO and increased antioxidant capacity in vital tissues of fish without having any impact on haematological and biochemical parameters, and on pituitary hormone gene expression. Statement of relevance We propose quercetin as supplement in fish diets.

This study investigated (1) the effects of cilostamide and follicular hemisections on in vitro oocyte meiotic resumption during a pre-maturation period of 12 h, (2) the levels of cAMP in bovine cumulus oocyte complexes (COCs) after pre-maturation, and (3) the expression of mRNA for CONNEXIN 43(CX43), HAS2, MATER, NPM2 and ZAR1 in denuded oocytes and cumulus cells from COCs that were subjected to both pre-maturation and maturation in vitro . To evaluate the effects of follicular hemisections and cilostamide on meiosis resumption, COCs were subjected to pre-IVM for 12 h in TCM-199 alone or in presence of 10 µM cilostamide, follicular hemisections and both cilostamide and follicular hemisections. After 6 h, COCs were used to measure the levels of cAMP, while after 12 h of pre-IVM, COCs were fixed to assess meiotic progression or stored to evaluate mRNA expression. At the end of this pre-IVM period, COCs were matured in vitro and meiotic progression was evaluated. The results showed that the presence of both follicular hemisections and cilostamide in pre-maturation medium increased significantly the percentages of oocytes at germinal vesicle stage (94%) when compared with COCs cultured in control medium alone or in presence of either follicular hemisections or cilostamide. After this pre-maturation period, 75% of oocytes reached metaphase II after 16 h of maturation, emphasizing that cilostamide did not have a toxic effect on oocytes. Moreover, oocytes cultured in medium containing cilostamide and follicular hemisections had significantly higher levels of cAMP when compared to other treatments. The presence of both cilostamide and follicular hemisections also increased the levels of mRNA expression for NPM2 and maintained those of MATER , ZAR1 , HAS2 and CX43 similar to control. In conclusion, cilostamide and follicular hemisections interact and promotes the maintenance of oocytes at germinal vesicle stage by increasing the levels cAMP in cultured COCs.

The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

The aim of this study was to evaluate the effect of the essential oil of Lippia alba (EOLA) as a feed additive on ionoregulatory and metabolic parameters and pituitary hormones expression in silver catfish, Rhamdia quelen , submitted to a stressful condition (stocking density of 10.6 kg m-3 and limited space). Fish were fed with different concentrations of EOLA (0.0 - control, 0.25 and 0.50 mL kg food-1) for 20 days. Metabolic parameters were not affected by the diet, with the exception of alanine aminotransferase, which was higher in the liver of fish fed 0.50 mL EOLA kg food-1. Plasma ions and activity of H+-ATPase did not change, but fish fed 0.25 mL EOLA kg food-1 presented higher Na+/K+-ATPase activity. Somatolactin expression in the pituitary was higher in the fish fed 0.25 mL EOLA kg food -1, but the expression of growth hormone and prolactin did not change. Therefore, dietary EOLA does not exert a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters. The use of 0.25 mL EOLA kg food-1 seems to be more suitable than 0.50 mL EOLA kg food-1 since the latter may be related to liver damage. RESUMO O objetivo deste estudo foi avaliar o efeito do óleo essencial de Lippiaalba (OELA) como aditivo em rações na ionoregulação, parâmetros metabólicos e expressão de hormônios hipofisários em jundiás, Rhamdiaquelen, submetidos a uma situação estressante (densidade de estocagem de 10,6 kg m-3 e espaço limitado). Os peixes foram alimentados com diferentes concentrações de OELA (0,0 - controle, 0,25 e 0,50 mL kg de ração-1) durante 20 dias. Parâmetros metabólicos não foram afetados pela dieta, com a exceção da alanina aminotransferase, que foi mais elevada no fígado dos peixes alimentados com 0,50 mL de OELA kg de ração-1. Íons plasmáticos e a atividade da H+-ATPase não apresentaram nenhuma alteração, mas os peixes alimentados com 0,25 mL OELA kg de ração-1 apresentaram maior atividade da Na+/K+-ATPase. A expressão da somatolactina na hipófise de peixes alimentados com 0,25 mL OELA kg de ração-1 aumentou, porém a expressão do hormônio de crescimento e da prolactina não mudou. Portanto, a adição do OELA na ração não tem um efeito protetor em jundiás submetidos a uma situação estressante, pois não influiu na maioria dos parâmetros medidos. O uso de 0,25 mL OELA kg de ração-1 parece ser mais adequado que 0,50 mL OELA kg de ração-1, uma vez que este nível de inclusão pode estar relacionado a danos hepáticos.

The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

Prostaglandin E2 (dinoprostone) is largely used for labor induction. However, one-third of patients do not respond to treatment. One cause of this poor response may be associated with changes in regulation of prostaglandin E receptors (EP1-4). In this study, we investigated EP mRNA expression in the uterine cervix and lower uterine segment myometrium for term births. Biopsies were obtained from women with successful (responders) and failed (non-responders) dinoprostone labor induction, while women that underwent spontaneous labor were included as controls. EP1 mRNA was upregulated in the cervical tissue of women who did not respond to dinoprostone induction. In addition, in the myometrium, significantly higher levels of EP3 mRNA were observed in women treated with dinoprostone, independent of their responsiveness. Dinoprostone-responders presented 3.6-fold higher levels of EP3 mRNA expression than the spontaneous labor group. Significantly higher levels of EP3 mRNA in the myometrium of the dinoprostone-treated group indicated that dinoprostone may regulate the EP3 gene on the transcriptional level. These results highlight the relationship between EP gene expression and delivery and indicate that understanding the regulation of prostaglandin E receptors may lead to improved labor induction.

The aim of this study was to assess the Mn toxicity to silver catfish considering Mn accumulation and oxidative status in different tissues, as well as pituitary hormone expression after acclimation to hypoxia. Silver catfish acclimated to hypoxia for 10 days and successively exposed to Mn (9.8 mg L−1) for an additional 10 days exhibited lower Mn accumulation in plasma, liver, kidneys and brain and prevented the hematocrit decrease observed in the normoxia group. Hypoxia acclimation also modified Mn-induced oxidative damage, which was observed by lower reactive species (RS) generation in gills and kidneys, decreased lipid peroxidation (LP) levels in gills, liver and kidneys and decreased protein carbonyl (PC) levels in liver, kidneys and brain. Manganese accumulation showed positive correlations with LP levels in gills and kidneys, as well as with PC levels in gills, liver and brain. In addition, hypoxia acclimation and Mn exposure increased catalase (CAT) activity in gills and kidneys and Na+/K+-ATPase activity in gills, liver and brain. Silver catfish that were acclimated under normoxia and exposed to Mn displayed increased pituitary prolactin (PRL) and decreased somatolactin (SL) expression. Interestingly, hypoxia acclimation prevented hormonal fluctuation of PRL and SL in fish exposed to Mn. These findings indicate that while the exposure of silver catfish to Mn under normoxia was related to metal accumulation and oxidative damage in tissues together with endocrine axis disruption, as represented by PRL and SL, hypoxia acclimation reduced waterborne Mn uptake, thereby minimizing oxidative damage and changes in hormonal profile. We hypothesized that moderate hypoxia is able to generate adaptive responses, which may be related to hormesis, thereby ameliorating Mn toxicity to silver catfish.

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.

Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (ION+Sr2+). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 μg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm3 and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm3 and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1,in vitromaturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 μM RAc or 0.5 μM RA. Presumptive zygotes and embryos (2–4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be producedin vitrofrom oocytes following protocols similar to those currently used for cattle.

Intratesticular injection (ITI) of sodium chloride (NaCl) is efficient for chemical castration of young calves, but its effects on calves welfare are unknown. Two experiments were conducted to evaluate the effects of ITI of 20% NaCl on stress and inflammatory markers in calves less than 20 days old and to assess the efficiency of ITI of 30% NaCl in 5 months old calves. In Experiment 1, control calves were only restrained and compared to calves submitted to castration through surgery (SC) and ITI with 20% NaCl (n = 9/group). No differences were observed for the eye corner temperature measured by thermography from 60 s before to 60 s after the procedures (P > 0.05). In the SC group, acute serum cortisol levels increased at 30 and 60 min after the procedure, but increased levels in the ITI group occurred only at 30 min (P 0.05). Scrotal temperature was higher at D1 in the SC group than for the other groups, but lowest at D4 compared to the control (both P < 0.05). In Experiment 2, histological sections of testes were compared after ITI with either 30% NaCl or 30% calcium chloride (CaCl2), to intact calves (control). After 60 days, intact seminiferous tubules and mediastinum were observed after ITI with 30% NaCl, whereas coagulative necrosis, inflammatory infiltration and calcification occurred after ITI with 30% CaCl2. Efficient chemical castration through ITI of 20% NaCl in young calves was followed by slight stress and inflammatory responses compared to surgical castration. However, ITI of 30% NaCl was ineffective for chemical castration of 5 months old calves.

In this study, a GnRH agonist, leuprolide acetate (LA), was given as a single depot injection before 48 h of life to Wistar female rats allotted to prenatal (E16-18) and postnatal androgenization (day 5 of life) by the use of testosterone propionate, looking for reproductive endpoints. Remarkably, a single injection of LA increased the estrus cycles in the postnatal group (PostN) from 0% to 25% of the estrus cycles in the postnatal LA treated group (PostN L). LA also reduced the serum testosterone levels and cysts and atretic follicles in PostN L in contrast with rats (>100 days) from the PostN group (p = 0.04). Prenatally androgenized rats (PreN) exhibited significant modifications in the hypothalamic genes, such as Gnrh. To the best of our knowledge, this is the first study to show that blockage of the GnRH axis with leuprolide acetate depot prevented the development of typical features (anovulation, cysts, atretic follicles) in a postnatal testosterone propionate rat model of PCOS.

Pregnancy diagnosis is an important tool for farm management. Ultrasonography is the main technique used for pregnancy diagnosis in ewes. As an alternative, radioimmunoassay (RIA) allows accurate and early detection of pregnancy-associated glycoproteins (PAGs) in sheep blood. However, radioactive-based techniques, as RIA, have been increasingly inadvisable due to environmental risk. Homology between ovine and bovine PAGs is high, and ELISA kits used for PAGs detection in cattle are safer than RIA. Thus, this study aimed to evaluate the feasibility of PAGs detection for pregnancy diagnosis in sheep serum samples using an ELISA kit produced for cattle. The sensitivity and specificity of the ELISA kit were 93.5% and 98.9%, respectively, whereas positive and negative predictive values were 99.0% and 93.1%, respectively, in comparison to ultrasonography diagnostic (control). PAGs reached consistently detectable concentrations in ovine serum around 33 days after mating. Accuracy of the ELISA test was 96.1% from 33 days of pregnancy until lambing. After parturition, PAGs were still detectable seven days post-lambing. However, from 21 days post-parturition, PAGs from the previous pregnancy were no longer detected in serum samples. In conclusion, the bovine ELISA kit can accurately detect pregnancy in sheep 33 days following mating, while PAGs levels from the previous gestation are no longer detected from 21 days post-partum. The evaluated ELISA test is a reliable tool for pregnancy diagnosis in sheep at random stages and as a complementary exam at early gestation.

O presente estudo teve como objetivo avaliar o efeito da Ang-(1-7) e de seu receptor (MAS) na regulação da ovulação. No experimento I, utilizando um modelo in vitro de cultivo de células foliculares, foi avaliado o efeito do tratamento com Ang-(1-7) ou do bloqueio do receptor MAS através do inibidor d-Ala7-Ang-(1-7) (A-779) na expressão de RNAm para epirregulina (Ereg; um marcador inicial do processo de ovulação) em células da granulosa. No experimento II, foi utilizado um modelo in vivo de injeção intrafolicular no qual vinte vacas tiveram o ciclo estral sincronizado e, quando os folículos atingiram um diâmetro mínimo de 12mm, foi realizada a injeção intrafolicular de A-779 ou solução salina 0,9%. No momento da injeção intrafolicular, foi realizada uma aplicação IM de análogo de GnRH. A suplementação com Ang-(1-7) ou o bloqueio de seu receptor MAS em sistema de cultivo de células da granulosa não alteraram o padrão de expressão de RNAm para Ereg. A aplicação intrafolicular de A-779 (10-5M) não bloqueou a ovulação quando realizada antes do início do pico esperado de LH (100% das vacas ovularam nos grupos A-779 e controle), sugerindo que a Ang-(1-7) não possui papel relevante no início da cascata ovulatória em bovinos.

Made available in DSpace on 2013-09-27T14:54:28Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T18:37:40Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:02Z No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Made available in DSpace on 2014-05-20T13:50:02Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P-4) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P-4 induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P-4 receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P-4 actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P-4-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P-4 in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P-4, providing further support to the AngII-P-4, sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P-4, and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation. (C) 2012 Elsevier B.V. All rights reserved. Universidade Federal de Santa Maria (UFSM), Lab Biotechnol & Anim Reprod BioRep, BR-97119900 Santa Maria, RS, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil

Angiotensin (Ang) II is widely known for its role in the control of systemic blood vessels. Moreover, Ang II acts on the vascular control of ovarian function, corpus luteum formation, and luteolysis. Over the past 10 years, our research group has been studying the new concept of the renin–angiotensin system (RAS) as an autocrine/paracrine factor regulating steroidogenesis and promoting different cellular responses in the ovary, beyond vascular function. We have developed and used differentin vivoandin vitroexperimental models to study the role of RAS in the ovary and a brief overview of our findings is presented here. It is widely accepted that there are marked species differences in RAS function in follicle development. Examples of species-specific functions of the RAS in the ovary include the involvement of Ang II in the regulation of follicle atresia in rats vs the requirement of this peptide for the dominant follicle development and ovulation in rabbits and cattle. More recently, Ang-(1–7), its receptor, and enzymes for its synthesis (ACE2, NEP, and PEP) were identified in bovine follicles, implying that Ang-(1–7) has an ovarian function. Other novel RAS components (e.g. (pro)renin receptor and renin-binding protein) recently identified in the bovine ovary show that ovarian RAS is poorly understood and more complex than previously thought. In the present review, we have highlighted the progress toward understanding the paracrine and autocrine control of ovarian antral follicle development and ovulation by ovarian tissue RAS, focusing onin vivostudies using cattle as a model.

Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE2, NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE2, NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased ( p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE2, NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE2, NEP and PEP were differentially expressed after GnRH treatment ( p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE2, NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Angiotensin II (AngII) has a role in ovarian follicle development, ovulation, and oocyte meiotic resumption. The objective of the present study was to characterise the AngII profile and the mRNA encoding RAS proteins in a bovine follicular wave. Cows were ovariectomised when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (day 2), slightly different (day 3), or markedly different (day 4). AngII was measured in the follicular fluid and the mRNA abundance of genes encoding angiotensin-converting enzyme (ACE), (pro)renin receptor, and renin-binding protein (RnBP) was evaluated in the follicular cells from F1 and F2. The AngII levels increased at the expected time of the follicular deviation in F1 but did not change in F2. However, the expression of the genes encoding ACE, (pro)renin receptor, and RnBP was not regulated in F1 but was upregulated during or after the follicular deviation in F2. Moreover, RnBP gene expression increased when the F1 was treated with the oestrogen receptor-antagonist in vivo. In conclusion, the AngII concentration increased in the follicular fluid of the dominant follicle during and after deviation and further supports our finding that RAS is present in the ovary regulating follicular dominance.

Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60–90 d postpartum, given 125 μg cloprostenol on two occassions, 12 h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5 mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24 h until Day 7 (Day 0 = treatment). When the largest follicle reached a designated diameter of 5–7, 8–10 or >10 mm, cows were randomly allocated to receive 2 mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100 mg of progesterone (P 4 ) given im, or an intravaginal device containing P 4 (3 × 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P 4 delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 μM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%;PP2, PGF2αor AngII was present in the co-culture system with follicular cells (PGE277.4%, PGF2α70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE2or PGF2αproduced by follicular cells.

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

Angiotensin II (AngII) is best known for its role in blood pressure regulation, but it also has documented actions in the reproductive system. There are two AngII receptors, type 1 (AGTR1) and type 2 (AGTR2). AGTR2 mediates the noncardiovascular effects of AngII and is expressed in the granulosa cell layer in rodents and is associated with follicle atresia. In contrast, expression of AGTR2 is reported to occur only in theca cells in cattle. The objective of the present study was to determine whether AngII also plays a role in follicle atresia in cattle. RT-PCR demonstrated AGTR2 mRNA in both granulosa and theca cells of bovine follicles. The presence of AGTR2 protein was confirmed by immunofluorescence. Abundance of AGTR2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas in theca cells, it did not change. Granulosa cells were cultured in serum-free medium, and treatment with hormones that increase estradiol secretion (FSH, IGF-I, and bone morphogenetic protein-7) increased AGTR2 mRNA and protein levels, whereas fibroblast growth factors inhibited estradiol secretion and AGTR2 protein levels. The addition of AngII or an AGTR2-specific agonist to granulosa cells in culture did not affect estradiol secretion or cell proliferation but inhibited abundance of mRNA encoding serine protease inhibitor E2, a protein involved in tissue remodeling. Because estradiol secretion is a major marker of nonatretic granulosa cells, these data suggest that AngII is not associated with follicle atresia in cattle but may have other specific roles during follicle growth.

There is evidence that the renin–angiotensin system plays an important role in ovulation in cattle. Using anin vivomodel, we investigated the role of angiotensin (Ang) II in bovine ovulation by injecting Ang II receptor antagonists into ovulatory follicles. Animals (n= 102) were pre-synchronized and, when the follicles reached 12 mm, they were given the respective treatment and the cows received GnRH agonist (i.m.) to induce ovulation. The ovulation rate was significantly lower when 100μM saralasin (Ang II receptor antagonist) was intrafollicularly injected (14.3%) in comparison with saline solution (83.3%). Based on these results, a second experiment was carried out to determine the timing of Ang II’s critical role in ovulation. Saralasin inhibited ovulation only when applied at 0 and 6 h (16.7 and 42.9% ovulation rate in the 0- and 6-h groups respectively), but not at 12 h (100%) following GnRH agonist treatment. To investigate the subtypes of Ang II receptors implicated in the LH-induced ovulation, losartan (LO; AT1-Ang II receptor antagonist), PD123 319 (AT2-Ang II receptor antagonist), LO+PD123 319, or saline were intrafollicularly injected when the cows were challenged with GnRH agonist. Ovulation was inhibited by PD123 319 and LO+PD123 319 (50.0 and 33.3% on ovulation rate respectively), but not by LO or saline solution (100% ovulation in both groups). From these results, we suggest that Ang II plays a pivotal role in the early mechanism of bovine ovulation via the AT2receptor subtype.

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (P

Subordinate follicles (SFs) of bovine follicular waves undergo atresia due to declining FSH concentrations; however, the signalling mechanisms have not been fully deciphered. We used an FSH-induced co-dominance model to determine the effect of FSH on signalling pathways in granulosa cells of the second-largest follicles (SF in control cows and co-dominant follicle (co-DF2) in FSH-treated cows). The SF was smaller than DF in control cows while diameters of co-DF1 and co-DF2 in FSH-treated cows were similar. The presence of cleaved CASP3 protein confirmed that granulosa cells of SFs, but not of DFs and co-DFs, were apoptotic. To determine the effect of FSH on molecular characteristics of the second-largest follicles, we generated relative variables for the second largest follicle in each cow. For this, variables of SF or co-DF2 were divided by the variables of the largest follicle DF or co-DF1 in each cow. There was higher transcript abundance of MAPK1/3 and AKT1/2/3 but lower abundance of phosphorylated MAPK3/1 in SF than co-DF2 granulosa cells. Abundance of mRNA and phosphorylated protein of STAT3 was higher in granulosa cells of control SF than FSH-treated co-DF2. SF granulosa cells had higher levels of LIFR and IL6ST transcripts, the two receptors involved in STAT3 activation. Further, lower transcript abundance of interleukin 6 receptor (IL6R), another receptor involved in STAT3 activation, indicated that STAT3 activation in SF granulosa cells could be mainly due to leukemia inhibitory factor (LIF) signalling. These results indicate that atresia due to lack of FSH is associated with activated LIF–STAT3 signalling in SF granulosa cells, as FSH treatment reversed such activation.

Introduction: This study was based on the hypothesis that some components of the angiotensin-(1-7) (Ang-(1-7)) system are differentially expressed during follicular development and can be involved in the follicular health/atresia transition in bovine. Material and methods: The largest (F1) and second largest follicles (F2) were collected from cows before (Day 2), dur- ing (Day 3), or after (Day 4) the expected moment of follicular deviation. In the second experiment, F1 was induced to atresia through intrafollicular injection of fulvestrant (estrogen receptor-antagonist) and, in both experiments, mRNA expression of the Mas receptor, ACE 2 , NEP, and PEP was evaluated in the granulosa and theca cells. Results: The mRNA expression of Mas receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation, while PEP mRNA increased during and after the deviation process. The mRNA expression of ACE 2 was upregulated in the granulosa cells of F1 during and after the follicular deviation. The mRNA expression of NEP was not regulated in F1 and F2. Mas receptor expression increased in the F1 induced to atresia. Conclusions: mRNA for Mas receptor, ACE 2 , and PEP are differentially expressed in granulosa cells throughout follicular development and the Mas receptor can be involved with the establishment of follicular dominance.

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.