Impact of hormonal modulation at proestrus on ovarian responses and uterine gene expression of suckled anestrous beef cows

Abstract

Background: This study evaluated the impact of hormonal modulation at the onset of proestrus on ovarian response and uterine gene expression of beef cows.

Methods: A total of 172 anestrous beef cows were assigned to one of four groups according to the treatment with estradiol cypionate (ECP) and/or equine chorionic gonadotropin (eCG) [CON (n = 43), ECP (n = 43), eCG (n = 44) and ECP + eCG (n = 42)].

Results: ECP-treated cows (ECP and ECP + eCG groups) presented greater occurrence of estrus (44.6% vs. 65.4%; P = 0.01) and pregnancy per AI [47.1% vs. 33.3%; P = 0.07], but similar progesterone (P4) concentration at subsequent diestrus than cows not treated with ECP (CON and eCG groups). Nonetheless, eCG-treated cows (eCG and ECP + eCG groups) presented larger follicle at timed AI (12.6 ± 0.3 vs. 13.5 ± 0.3 mm; P = 0.03), greater ovulation rate (96.5% vs. 82.6%; P = 0.008) and greater P4 concentration at d 6 (3.9 ± 0.2 vs. 4.8 ± 0.2 ng/mL; P = 0.001) than cows not treated with eCG (CON and ECP groups). Next, cows with a new corpus luteum 6 d after TAI were submitted to uterine biopsy procedure. Uterine fragments [CON (n = 6), ECP (n = 6)] were analyzed by RNA-Seq and a total of 135 transcripts were differentially expressed between groups (73 genes up-regulated by ECP treatment). Subsequently, uterine samples were analyzed by qPCR (genes associated with cell proliferation). ECP treatment induced greater abundance of PTCH2 (P = 0.07) and COL4A1 (P = 0.02), whereas suppressed EGFR (P = 0.09) expression. Conversely, eCG treatment increased abundance of HB-EGF (P = 0.06), ESR2 (P = 0.09), and ITGB3 (P = 0.05), whereas it reduced transcription of ESR1 (P = 0.05). Collectively, supplementation with ECP or eCG at the onset of proestrous of anestrous beef cows influenced ovarian responses, global and specific endometrial gene expression.

Conclusion: Proestrus estradiol regulate the endometrial transcriptome, particularly stimulating proliferative activity in the endometrium.

Keywords: Cattle; Endometrium; Estradiol; Transcriptome; eCG.

Fig. 1

Schematic diagram of the synchronization of ovulation protocol in suckled anestrous beef cows. EB = 2 mg of estradiol benzoate; P4 = progesterone; P4 insert = previously used intravaginal P4 insert containing 1.0 g of P4; GnRH = 100 mg of gonadorelin; ECP = 1 mg of estradiol cypionate; eCG = 400 IU of equine chorionic gonadotropin; PGF2α = 0.25 mg of cloprostenol; US = ultrasound examination; BS = blood sample. Cows from ECP group received ECP and cows from eCG group received eCG, while cows from CON did not receive any further treatment and cows from ECP + ECG received both treatments

Fig. 2

Volcano plot obtained from DESeq analysis. Volcano plot shows that the vertical lines axe is log₂-fold change and the horizontal axis is the statistical significance (P value ≤ 0.10). Genes with P value ≤0.10 are marked with blue dots

Fig. 3

Heat map obtained from DESeq2 analysis. Each column represent one sample showing the intensity of expression profile per gene. The colors in the map display the relative standing of the reads count data; GREEN indicates a count value that is lower than the mean value of the row while red indicates higher than the mean. The shades of the color indicate distance from each data point to the mean value of the row

Fig. 4

Comparison of gene expression between suckled anestrous beef cows receiving 1 mg of estradiol cipionate (ECP) and/or 400 IU of equine chorionic gonadotropin (eCG) at onset of the proestrus [CON (n = 11), ECP (n = 11), eCG (n = 12) and ECP + ECG (n = 11)]. The amounts of ESR1, ESR2, PGR, and PTCH2 transcripts are expressed in relation to control (CON) untreated cows. Expression values were normalized by the geometric mean of GAPDH, ACTB, and RPS18. The P values refer to comparisons made for each gene between groups (effects of ECP, eCG and interaction between ECP and eCG)

Fig. 5

Comparison of gene expression between suckled anestrous beef cows receiving 1 mg of estradiol cipionate (ECP) and/or 400 IU of equine chorionic gonadotropin (eCG) at onset of the proestrus [CON (n = 11), ECP (n = 11), eCG (n = 12) and ECP + ECG (n = 11)]. The amounts of EGFR, HBEGF, ITGB3, and COL4A1 transcripts are expressed in relation to control (CON) untreated cows. Expression values were normalized by the geometric mean of GAPDH, ACTB, and RPS18. The P values refer to comparisons made for each gene between groups (effects of ECP, eCG and interaction between ECP and eCG)