Sperm utilize tumor necrosis factor alpha to shut down a 2-methoxyestradiol nongenomic pathway that accelerates oviductal egg transport in the rat.

In brief: Mating shuts down the 2-methoxyestradiol (2ME) nongenomic pathway that accelerates oviductal egg transport in the rat. This study shows that sperm cells, but not vaginocervical stimulation, utilize TNF-α to shut down this 2ME nongenomic pathway. Abstract: The transport of oocytes or embryos throughout the oviduct to the implantation site in the uterus is defined as egg transport. In the rat, 2-methoxyestradiol (2ME) accelerates egg transport through the oviduct via a nongenomic pathway. Mating is known to shut down this 2ME pathway and then trigger an estradiol genomic pathway that accelerates egg transport. Here, we tested whether intrauterine insemination (IUI) or vaginocervical stimulation (VCS) shuts down the 2ME nongenomic pathway that accelerates egg transport, and if these mating components require tumor necrosis factor alpha (TNF-α). Levels of TNF-α and the mRNA for TNF-α receptors were measured in the oviduct of IUI or VCS rats. The tissue distribution of TNF-α receptor proteins and the concentration of the mRNA for catechol-O-methyl transferase (Comt) and 2ME were also analyzed in the oviduct. Finally, we assessed whether 2ME accelerates egg transport in IUI or VCS rats previously treated with the TNF-α antagonist W9P9QY. Results show that IUI, but not VCS, increased TNF-α and their receptors in the oviduct. IUI and VCS did not change the tissue distribution of TNF-α receptors; however, both decreased the oviductal concentration of Comt and 2ME. IUI and VCS each blocked the 2ME-induced egg transport acceleration; however, only the IUI was antagonized by the TNF-α antagonist. We concluded that IUI and VCS inhibit the 2ME nongenomic pathway that accelerates egg transport; however, the vias of action are distinct, with a TNF-α increase on spermatozoa presence being required for the shutdown of the 2ME pathway.

Prolactin gene expression in the pituitary of rats subjected to vaginocervical stimulation requires Erk-1/2 signaling.

Vaginocervical stimulation (VCS) induces twice-daily prolactin (PRL) surges resulting in pseudopregnancy in the rat. Furthermore, activation of the extracellular signal-regulated kinase-1/2 (Erk-1/2) is involved in the effect of estradiol (E2) on the Prl gene expression in pituitary cells. Herein, we investigated whether Erk-1/2 signaling is involved in the control of Prl expression in the pituitary of VCS rats and whether VCS regulates the effect of E2 on Erk-1/2 and Prl in the pituitary. Estrous rats were assigned as control or VCS groups and 0, 6, 12 or 24h later the levels and localization of phosphorylated Erk-1/2 (p-Erk-1/2) were analyzed in the pituitary. The effect of an Erk-1/2 inhibitor PD98059 on the Prl level in the pituitary of control or VCS rats was also analyzed. Other control or VCS rats were treated with E2 and the level of p-Erk-1/2 and Prl were measured in the pituitary. In control rats, p-Erk-1/2 decreased at 6 and 12h and increased at 24h while Erk-1/2 was phosphorylated at all time points in VCS rats. p-Erk-1/2 was localized only in the anterior pituitary. PD98059 decreased Prl level in VCS, but not in control rats. Estradiol decreased Erk-1/2 phosphorylation although did not change Prl level in the pituitary of control or VCS rats. These findings show that prolonged activation of Erk-1/2 is necessary to induce Prl expression in the pituitary of VCS rats; however, VCS does not influence the role of E2 on the activation of Erk-1/2 and Prl expression the pituitary.

The role of mating in oviduct biology.

The oviduct connects the ovary to the uterus, and is subject to changes that influence gamete transport, fertilization, and early embryo development. The ovarian steroids estradiol and progesterone are largely responsible for regulating oviduct function, although mating signals also affect the female reproductive tract, both indirectly, through sensory stimulation, and directly, through contact with seminal plasma or spermatozoa. The resulting alterations in gene and protein expression help establish a microenvironment that is appropriate for sperm storage and selection, embryo development, and gamete transport. Mating may also induce the switch from a non-genomic to a genomic pathway of estradiol-accelerated oviduct egg transport, reflecting a novel example of the functional plasticity in well-differentiated cells. This review highlights the physiological relevance of various aspects of mating to oviduct biology and reproductive success. Expanding our knowledge of the mating-associated molecular and cellular events in oviduct cells would undoubtedly facilitate new therapeutic strategies to treat infertility attributable to oviduct pathologies. Mol. Reprod. Dev. 83: 875-883, 2016 © 2016 Wiley Periodicals, Inc.

Estradiol increases IP3 by a nongenomic mechanism in the smooth muscle cells from the rat oviduct.

Estradiol (E2) accelerates egg transport by a nongenomic action, requiring activation of estrogen receptor (ER) and successive cAMP and IP3 production in the rat oviduct. Furthermore, E2 increases IP3 production in primary cultures of oviductal smooth muscle cells. As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E2 in the oviduct, herein we determined the mechanism by which E2 increases IP3 in these cells. Inhibition of protein synthesis by Actinomycin D did not affect the E2-induced IP3 increase, although this was blocked by the ER antagonist ICI182780 and the inhibitor of phospholipase C (PLC) ET-18-OCH3. Immunoelectron microscopy for ESR1 or ESR2 showed that these receptors were associated with the plasma membrane, indicating compatible localization with E2 nongenomic actions in the smooth muscle cells. Furthermore, ESR1 but not ESR2 agonist mimicked the effect of E2 on the IP3 level. Finally, E2 stimulated the activity of a protein associated with the contractile tone, calcium/calmodulin-dependent protein kinase II (CaMKII), in the smooth muscle cells. We conclude that E2 increases IP3 by a nongenomic action operated by ESR1 and that involves the activation of PLC in the smooth muscle cells of the rat oviduct. This E2 effect is associated with CaMKII activation in the smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity necessary to accelerate oviductal egg transport.

Mating decreases plasma levels of TGFß1 and regulates myosalpinx expression of TGFß1/TGFBR3 in the rat.

Mating shuts down a 2-methoxyestradiol (2ME)-dependent, non-genomic activity that is responsible for accelerating egg transport in the rat oviduct. The aims of this work were to investigate the role of TGFß1 in this 2ME-reduced activity and to determine the effect of mating on the expression and distribution of TGFß1 and its receptor TGFBR3 in the rat oviduct. We determined the level of TGFß1 in the plasma and oviductal fluid at 1, 3, or 6 hr during Day 1 of the oestrous cycle in unmated or mated animals. We then examined if 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a neutralizing TGFß1 antibody. The expression of Tgfb1 and Tgfbr3 mRNA and the level and distribution of TGFBR3 protein in the oviduct were also determined at these time points. Mating decreased TGFß1 in the plasma, but not in the oviductal fluid, whereas antibody neutralization of circulating TGFß1 did not prevent the effect of 2ME on egg transport. Mating decreased Tgfb1 and hastened the increase in TGFBR3 abundance in the myosalpinx. These results indicate that mating decreased circulating levels of TGFß1 without shutting down the non-genomic 2ME response that normally accelerates egg transport. Levels of Tgfb1 transcript and TGFBR3 protein, however, changed in the myosalpinx of mated rats, suggesting a role for mating-associated factors in the autocrine and paracrine effects of TGFß in the oviduct.

Estradiol increases cAMP in the oviductal secretory cells through a nongenomic mechanism.

In the rat oviduct, estradiol (E2) accelerates egg transport by a nongenomic action that requires previous conversion of E2 to methoxyestrogens via catechol-O-methyltranferase (COMT) and activation of estrogen receptor (ER) with subsequent production of cAMP and inositol triphosphate (IP3). However, the role of the different oviductal cellular phenotypes on this E2 nongenomic pathway remains undetermined. The aim of this study was to investigate the effect of E2 on the levels of cAMP and IP3 in primary cultures of secretory and smooth muscle cells from rat oviducts and determine the mechanism by which E2 increases cAMP in the secretory cells. In the secretory cells, E2 increased cAMP but not IP3, while in the smooth muscle cells E2 decreased cAMP and increased IP3. Suppression of protein synthesis by actinomycin D did not prevent the E2-induced cAMP increase, but this was blocked by the ER antagonist ICI 182 780 and the inhibitors of COMT OR 486, G protein-α inhibitory (Gαi) protein pertussis toxin and adenylyl cyclase (AC) SQ 22536. Expression of the mRNA for the enzymes that metabolizes estrogens, Comt, Cyp1a1, and Cyp1b1 was found in the secretory cells, but this was not affected by E2. Finally, confocal immunofluorescence analysis showed that E2 induced colocalization between ESR1 (ERα) and Gαi in extranuclear regions of the secretory cells. We conclude that E2 differentially regulates cAMP and IP3 in the secretory and smooth muscle cells of the rat oviduct. In the secretory cells, E2 increases cAMP via a nongenomic action that requires activation of COMT and ER, coupling between ESR1 and Gαi, and stimulation of AC.

Changes in the gene expression pattern induced by 2-methoxyestradiol in the mouse uterus.

2-Methoxyestradiol (2ME) is an estrogen metabolite with antitumor and antiangiogenic properties, although their effects on the reproductive tissues are not well-determined. Furthermore, it is not very clear whether 2ME is part of the intracellular signaling of estradiol (E2) or it acts through other signaling pathways. The purpose of this study was to determine changes in the gene expression pattern in the mouse female reproductive tract induced by 2ME, under conditions in which this metabolite has no estrogenic activity. Therefore, we first compared the effect of 2ME or E2 on the uterine weight and epithelial cell height, and on the ovarian weight and the number of follicles of immature mice. Then, we examined the gene expression profile in the uterus of immature mice treated with 2ME or E2 and we selected three genes scd2, snx6, and spon1, to confirm differential regulation by E2 and 2ME in the uterine cells using real-time PCR. Finally, in order to explore the physiologic relevance of the 2ME-induced genes we determined the expression and localization of the F-spondin protein encoded by spon1 in the uterus of mature mice treated with E2 or 2ME. Estradiol and 2ME reduced the ovarian weight and decreased the number of follicles ≥ 300 µm, whereas E2 increased the uterine weight and epithelial cell height but not 2ME, indicating that 2ME did not have estrogenic activity in the mouse uterus. Microarray analysis showed that 1.8 % of the uterine genes were regulated by E2 and 0.23 % by 2ME, while 0.04 % was regulated by E2 and 2ME. The mRNA for scd2 was exclusively increased by 2ME, whereas snx6 and spon1 were up-regulated by E2 and 2ME, but the response to 2ME was more intense. F-spondin was mainly expressed in the uterine stroma layer although 2ME or E2 did not change its localization in the uterine cells. We conclude that 2ME regulates a group of genes in the mice uterus, independently of estrogenic activity, suggesting a functional involvement of 2ME in the mammalian uterus.

Tumour necrosis factor-α is the signal induced by mating to shutdown a 2-methoxyestradiol nongenomic action necessary to accelerate oviductal egg transport in the rat.

Mating shut down a 2-methoxyestradiol (2ME) nongenomic action necessary to accelerate egg transport in the rat oviduct. Herein, we investigated whether tumour necrosis factor-α (TNF-α) participates in this mating effect. In unmated and mated rats, we determined the concentration of TNF-α in the oviductal fluid and the level of the mRNA for Tnf-a (Tnf) and their receptors Tnfrsf1a and Tnfrsf1b in the oviduct tissues. The distribution of the TNFRSF1A and TNFRSF1B proteins in the oviduct of unmated and mated was also assessed. Finally, we examined whether 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a rat recombinant TNF-α alone or concomitant with a selective inhibitor of the NF-κB activity. Mating increased TNF-α in the oviductal fluid, but Tnf transcript was not detected in the oviduct. The mRNA for TNF-α receptors as well as their distribution was not affected by mating, although they were mainly localized in the endosalpinx. Administration of TNF-α into the oviduct of unmated rats prevented the effect of 2ME on egg transport. However, the NF-κB activity inhibitor did not revert this effect of TNF-α. These results indicate that mating increased TNF-α in the oviductal fluid, although this not associated with changes in the expression and localization of TNF-α receptors in the oviductal cells. Furthermore, TNF-α mimicked the effect of mating on the 2ME-induced egg transport acceleration, independently of the activation of NF-κB in the oviduct. We concluded that TNF-α is the signal induced by mating to shut down a 2ME nongenomic action in the rat oviduct.