Uterine secretome and its modulation in rat (Rattus norvegicus)

Abstract

The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (nZ17) and metestrous (nZ18) phases and also from pregnant (nZ17) and pseudopregnant (nZ17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography-tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P!0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P!0.05; t-test) abundance of b-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIa), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P!0.05; t-test) levels of UF b-actin and Rho-GDIa. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.

Cytokines and Blastocyst Hatching

“…Even more, the involvement of cytokines in modulating hatching‐associated or hatching‐specific proteases is virtually not known. Of relevance in this context is the fact that a plethora of secretory factors, including cytokines, are present in the endometrium‐derived uterine luminal fluid which could regulate blastocyst development and function . However, there is no evidence to show that uterine luminal fluid cytokines are involved in the hatching process in vivo .…”

Membrane‐initiated estrogen signaling in prostate cancer: A route to epithelial‐to‐mesenchymal transition

“…Spots corresponding to the proteins displaying differential phosphorylation, as revealed by immunodetection with pan‐phospho antibody and subsequent alignment of luminograms and 2D blots), were manually excised from duplicate silver‐stained gels. Destained gel plugs were digested with trypsin as described previously . Peptides were extracted from the gel plugs by incubation in 50% acetonitrile (ACN) solution containing 0.1% Trifluoroacetic acid (TFA) for 15 minutes.…”

“…Destained gel plugs were digested with trypsin as described previously. 26 Peptides were extracted from the gel plugs by incubation in 50% acetonitrile (ACN) solution containing 0.1% Trifluoroacetic acid (TFA) for 15 minutes. Lyophilized peptides were reconstituted in 50% ACN solution with 0.1% TFA for MALDI TOF-TOF analysis.…”

Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells

“…Destained gel plugs were digested with 0.01 mg/ml sequencing‐grade trypsin (Sigma–Aldrich) for 16 hr at 37°C. Tryptic peptides were extracted from gel plugs using the protocol described previously (Bhutada et al, ). Vacuum‐dried peptides were reconstituted in sample diluent (30% v/v acetonitrile, 0.1% trifluoroacetic acid) and mixed with an equal volume of matrix (a‐cyano‐4‐hydroxycinnamic acid, 70% v/v acetonitrile, 1% trifluoroacetic acid), spotted in duplicate on a target plate, and air‐dried.…”