Search

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.

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.

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.

The anti-Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p0.05), but they tended to be greater in DFs than SFs (p0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p0.05) between both co-dominant follicles collected from the FSH-treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co-dominant follicles.

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.

Summary The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT-PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p

Background: Estradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells. Methods: Granulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes. Results: ESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes. Conclusions: Our findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control—surgically castrated immediately after birth; positive control —intact males; G1—ITI from 1- to 5-day old; G2—ITI from 15- to 20-day old; and G3—ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P

Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia.

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E2) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7–8 mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3 mm for 0, 0.1, and 1 μg/ml FGF10, respectively, at 72 h after treatment; PLHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E2 production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.