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. Reproduction (2015) 150 395-403
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<0.05), in which no testicular structure and sperm cells were observed during breeding soundness evaluation. Rectal and scrotal temperatures were not affected by different procedures. In conclusion, ITI of hypertonic NaCl solution induces sterility and completely suppresses testosterone secretion when performed during the first 20 days of life.
BackgroundEstradiol (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.MethodsGranulosa 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.ResultsESR1 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.ConclusionsOur 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.
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