Category: Pregnancy

DISCUSSION(3)

In the present study, marked differences in UTMP mRNA expression were apparent in sGE between Days 17 and 50 of pregnancy, with higher levels of UTMP mRNA in the upper sGE compared to the lower sGE near the myometrium. This dichotomy in expression may be related to the fact that deeper glands of the intercaruncular endometrium undergo proliferation during early pregnancy, which precedes maximal levels of protein synthesis by the intercaruncular endometrium. Indeed, intrauterine administration of recombinant oPL specifically stimulates proliferation of deep sGE. Likewise, GE proliferation occurs between Days 17 and 50 of pregnancy. Once fully developed, uterine glands enlarge between Days 55 and 80, a process presumably stimulated by PRL, PL, and placental GH. In support of this concept, all endometrial sGE expressed abundant levels of UTMP mRNA after Day 50 of pregnancy, which correlates with large amounts of PL secreted by the placenta and appearance of PL in maternal serum. In addition, temporal changes in PL production by the conceptus closely parallelled changes in total protein synthesized and secreted by the intercaruncular endometrium during pregnancy.

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In the present study, overall changes in steady-state levels of intercaruncular endometrial UTMP mRNA during pregnancy closely paralleled changes in PL production by the placenta and release of newly synthesized proteins, including UTMP, by intercaruncular endometrial explants from pregnant ewes. Spencer et al. reported that intrauterine administration of recombinant oPL increased proliferation of sGE and enhanced UTMP and OPN expression in the sGE; they suggested that PL acts in a paracrine manner on PRL-R-positive sGE to increase UTMP gene expression and perhaps the transport and/or synthesis of other GE-specific secretory products.

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DISCUSSION(1)

Expression of PRL-R by uterine endometrium has been reported for the cow, human, pig, rat , rabbit, and ewe. As in the cyclic human uterus, pRL-R expression was expressed specifically in the endometrial GE. Results from this study and others extend those of Cassy et al. in sheep and Jones et al. in the human by suggesting that lactogenic hormones play key roles in regulation of GE function. The present study showed that the initial increase in expression of the PRL-R gene in sGE occurs between Days 17 and 19. This period coincides with the time of onset of PL secretion by trophectoderm and down-regulation of progesterone receptor (PR) expression in sGE. Effects of PL on PRL-R gene expression in the endometrium are not known, but Galsgaard et al. found that both PRL and GH increased PRL-R gene expression in insulin-producing cells.

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ImmunohistochemicalAnalysis of PRL-R Protein in the Ovine Uterus The location of PRL-R protein in the cyclic and pregnant ovine uteri from study 1 was assessed using immunofluorescence antibody analysis of frozen uterine sections (Fig. 5). Immunoreactive PRL-R protein was present exclusively in the apical aspect of the endometrial sGE of both cyclic and pregnant uteri. As expected from in situ hybridization results, no specific signal was detected in endometrial LE, superficial GE, stroma, or myometrium. In pregnant ewes, no specific signal was detected in the chorioallantoic placenta (data not shown). Negative controls, in which normal rabbit IgG was substituted for rabbit anti-bovine PRL-R IgG, showed no specific staining in endometrial glands. Steady-State Levels of Endometrial UTMP mRNA Steady-state levels of UTMP mRNA in the intercarun-cular endometrium were affected (P < 0.01, cubic) by day of pregnancy (Fig. 6). Endometrial levels of UTMP mRNA increased approximately 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined to Day 120. …Read the rest of this article

RESULTS(2)

As shown in Figure 2B, both long (310 bp) and short (207 bp) forms of the ovine PRL-R mRNA were present in the intercaruncular endometrium of Day 20 to 120 pregnant ewes from study 2. Two specific PCR products (310 bp and 350 bp) were consistently detected using the long PRL-R mRNA primers. Sequence analysis of the two long PRL-R PCR products indicated that the 310-bp product was identical to the long ovine PRL-R cDNA, whereas sequence analysis of the unexpected 350-bp cDNA product derived from the long PRL-R primers revealed that the cDNA included the 39-bp exon of the short PRL-R mRNA. The 207-bp product generated by the short PRL-R primers was found to be the short PRL-R mRNA and included the 39-bp alternatively spliced exon. The ratio of the 350-bp to 310-bp long PRL-R cDNA products did not change with day of pregnancy (P > 0.10). Further, the relative ratio of short to long PRL-R mRNAs revealed no significant effect of day (P > 0.10).

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Steady-State Levels of Endometrial PRL-R mRNA

Steady-state levels of PRL-R mRNA in the endometrium were affected by day of the estrous cycle and pregnancy (Fig. 1). In cyclic ewes, endometrial PRL-R mRNA levels were affected (P < 0.09, cubic) by day, but changes were not dramatic (Fig. 1A). Between Days 11 and 19 of pregnancy (study 1), endometrial PRL-R mRNA increased (P < 0.01, quadratic) almost 2-fold, with the largest increase between Days 17 and 19 (Fig. 1A). On Days 11-15, endometrial PRL-R mRNA levels were not different between cyclic and pregnant ewes (P > 0.10, day X status). In study 2, slot-blot hybridization analyses indicated that steady-state levels of PRL-R mRNA in the intercaruncular endometrium increased linearly (P < 0.01) from Days 20 to 120 of pregnancy (Fig. 1B).

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MATERIALS AND METHODS(5)

Immunocytochemical Analysis

Frozen sections (4-8 ^m) of uterine tissues embedded in OCT compound in study 1 were cut with a cryotome (Lipshaw Manufacturing, Detroit, MI) and mounted on Superfrost/Plus microscope slides (Fisher Scientific). Concep-tuses were present in the uterine lumen of sections from Day 19 pregnant ewes, as these uteri were not flushed prior to uterine tissue collection. Sections were fixed in -20°C methanol for 10 min, permeabilized with 0.3% Tween 20 in 0.02 M PBS, and then blocked in antibody dilution buffer (two parts 0.02 M PBS, 1.0% BSA, 0.3% Tween 20 [pH 8.0] and one part glycerol) containing 5% normal goat serum for 1 h at room temperature. Sections were rinsed in PBS and incubated overnight at 4°C with 20 ^g/ml rabbit anti-bovine PRL-R IgG or 20 ^g/ml normal rabbit IgG (Sigma, St. Louis, MO) as a control. After three rinses in PBS for 10 min each, sections were incubated with fluorescein-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA) for 1 h at room temperature and again washed in PBS three times for 10 min each. Sections were then overlaid with a coverslip and Prolong Antifade mounting reagent (Molecular Probes, Eugene, OR) and viewed with a Zeiss Photomicroscope III (Carl Zeiss, Thornwood, NJ) equipped with a fluorescein isothiocyanate filter set.

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