Evidence of endometrial amino acid metabolism and transport modulation by peri-ovulatory endocrine profiles driving uterine receptivity

Background: In beef cattle, changes in the periovulatory endocrine milieu are associated with fertility and conceptus growth. A large preovulatory follicle(POF) and the resulting elevated concentrations of progesterone(P4) during diestrus positively affect pregnancy rates. Amino acids(AA) are important components of maternally derived secretions that are crucial for embryonic survival before implantation. The hypothesis is that the size of the POF and the concentration of P4 in early diestrus modulate the endometrial abundance of SLC transcripts related to AA transport and metabolism and subsequently impact luminal concentrations of AA. The follicle growth of Nelore cows was manipulated to produce two experimental groups: large POF and CL(LF-LCL group) and small POF and CL(SF-SCL group). On Day 4(D4; Experiment 1) and Day 7(D7; Experiment 2) after Gn RH-induced ovulation(Gn RH treatment = D0), the animals were slaughtered and uterine tissues and uterine washings were collected. q RT-PCR was used to evaluate the expression levels of AA transporters in D4 and D7 endometrial tissues.The concentrations of AA were quantified in D4 and D7 uterine washings by HPLC.Results: Transcript results show that, on D4, SLC6 A6, SLC7 A4, SLC17 A5, SLC38 A1, SLC38 A7 and SCLY and on D7 SLC1 A4, SLC6 A1, SLC6 A14, SLC7 A4, SLC7 A7, SLC7 A8, SLC17 A5, SLC38 A1, SLC38 A7, SLC43 A2 and DDO were more abundant in the endometria of cows from the LF-LCL group(P < 0.05). In addition, concentrations of AA in the uterine lumen were influenced by the endocrine profiles of the mother. In this context, D4 uterine washings revealed that greater concentrations of taurine, alanine and α-aminobutyric acid were present in SF-SCL(P < 0.05).In contrast, lower concentrations of valine and cystathionine were quantified on D7 uterine washings from SF-SCL cows(P < 0.05).Conclusion: The present study revealed an association between the abundance of transcripts related to AA transport and metabolism in the endometrium and specific periovulatory endocrine profiles related to the receptive status of the mother. Such insights suggest that AAs are involved in uterine function to support embryo development.

Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle

Background: A major,unresolved issue is how the uterine microenvironment determines pregnancy success in cattle.Before implantation,conceptus development depends on the uterine secretome(i.e.,histotroph).Despite its pivotal role,little is known about the dynamics of histotroph synthesis and changes in composition throughout the early diestrus and the relevance to pregnancy establishment.We hypothesize that disturbances on histotroph composition affect the establishment of pregnancy.Aim was to disturb histotroph composition at early diestrus and verify the effects on:(Exp.1) timing to restore its composition; and(Exp.2) pregnancy rate after multiple-embryo transfer.Estrous cycle of multiparous Nelore cows were synchronized and estrus was considered d 0(D0) of the experiments.Disturbance was through flushing each uterine horn with 30 m L of DMPBS and collecting the resulting uterine luminal flushing(ULF) on D1; D4; D7; D1 + D4 + D7.Control group remained not-collected.In Exp.1,ULF was collected on D7.5 from all animals and used for quantification of total protein concentration and abundance of albumin.In Exp.2,three in vitro-produced embryos were transferred to the uterine horn ipsilateral to the ovary containing the CL on D7.5 and pregnancy was checked on D25 by ultrasound.Results: In Exp.1,ULF collection on D4 or D7 increased(1.5-to 2.2-folds) the total protein concentration and albumin abundance.ULF collection on D1 did not alter(P > 0.10) these endpoints.In Exp.2,ULF collected on D4 or D7 decreased pregnancy rates to approximately half of that measured in the remaining groups.Conclusions: Subtle perturbations imposed to the native intrauterine milieu,such as those caused by a single,low-volume collection of ULF,profoundly disturbs intrauterine composition and pregnancy success.At least 4 d were necessary for the uterus to recover its composition and the functional capacity to carry post-implantation gestation.

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

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 q PCR（genes associated with cell proliferation）. ECP treatment induced greater abundance of PTCH2（P = 0.07） and COL4 A1（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.