Temporal regulation of fibroblast growth factors and their receptors in the endometrium and conceptus during the pre-implantation period of pregnancy in cattle.

We hypothesised that the expression pattern of members of the fibroblast growth factor (FGF) family would be altered in the endometrium as the oestrous cycle/early pregnancy progressed associated with changes in the expression pattern of their receptors in the developing embryo/conceptus. Expression of FGF1 and FGF10 transcript variants 1 and 2 increased significantly as the oestrous cycle/early pregnancy progressed. Neither progesterone (P4) supplementation nor pregnancy status significantly affected the expression of any of the FGF ligands studied. However, there was a significant interaction between day, pregnancy and P4 status on FGF2 expression (P<0.05) and a significant interaction between P4 status and day on FGF10_tv2 expression. FGF10 protein was localised in the luminal and glandular epithelium as well as the stroma but was not detected in the myometrium. By RNA sequencing, the expression of FGF ligands in the developing embryo/conceptus was found to be minimal. The expression of FGF receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, FGFRL1 and FRS3 was significantly affected by the stage of conceptus development. Interestingly, the expression of FGFR1 and FGFR4 was higher during early embryo development (days 7-13, P<0.05) but decreased on day 16 (P<0.05) while FGFR2 (P<0.001) expression was similar from day 7 through to day 13, with a significant increase by day 16 (P<0.05) that was maintained until day 19 (P>0.05). In conclusion, these data demonstrate that FGF ligands are primarily expressed by the endometrium and their modulation throughout the luteal phase of the oestrous cycle/early pregnancy are associated with alterations in the expression of their receptors in the embryo/conceptus.

Estrogen-induced maldevelopment of the penis involves down-regulation of myosin heavy chain 11 (MYH11) expression, a biomarker for smooth muscle cell differentiation.

Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%-80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect.

Exposure of neonatal rats to anti-androgens induces penile mal-developments and infertility comparable to those induced by oestrogens.

We previously reported that oestrogen exposure in neonatal rats induced permanent infertility and malformed penis characterized by fat accumulation, which replaced most of the smooth muscle cells and cavernous spaces in the body of the penis, structures essential for erection. The objective of this study was to determine if reduced androgen production/action in the neonatal period, in the absence of exogenous oestrogen exposure, induces penile deformities similar to those caused by oestrogen. Male rats were treated from postnatal days 1-6 with GnRH antagonist antide (A, 10 mg/kg) or androgen receptor (AR) antagonist flutamide (F, 50 mg/kg) or F + A, with or without AR agonist dihydrotestosterone (DHT, 20 mg/kg). For comparison, pups received diethylstilbestrol (DES, 0.1 mg/kg), with or without DHT. Tissues were collected at ages 7 and 12 days and at adulthood. Flutamide alone decreased penile length and weight significantly (p < 0.05), but it caused neither fat accumulation, nor affected fertility (80% vs. 87% in controls). Antide alone reduced penile length and weight significantly, and induced fat accumulation in 4/11 rats and infertility in 13/14 rats. Conversely, all 11 F + A-treated rats, similar to all nine DES-treated rats, had fat accumulation and loss of smooth muscle cells and cavernous spaces in the body of the penis and were infertile. In addition, reductions in penile length and weight were higher than in rats treated with F or A alone. DHT co-administration mitigated penile deformities in the DES group, but did not in the F + A group. Testicular testosterone was reduced by 70-95% at 7 or 12 days of age in all treated groups, except in the F group, which had threefold higher testosterone than controls. Collectively, data unequivocally show that reduced androgen production/action in the neonatal period, in the absence of oestrogen exposure, induces permanent infertility and malformed penis similar to that caused by oestrogen.

Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle.

Progesterone-induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n = 140) or not (n = 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P < 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P > 0.05). LGALS9 and LGALS3BP were expressed at low levels in both cyclic and pregnant endometria until day 13. On day 16, expression increased only in the pregnant heifers (P < 0.0001). LGALS1 and LGALS3 decreased on day 7 (P < 0.0001) and remained low until day 16. Pregnancy had no effect on the expression of LGALS1, LGALS3, and PIBF. Additionally, LGALS9 and PIBF proteins were expressed in distinct uterine cell types. These results indicate that the galectins may be involved in uterine receptivity and/or implantation in heifers.

Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

The effect of elevated progesterone and pregnancy status on mRNA expression and localisation of progesterone and oestrogen receptors in the bovine uterus.

To investigate the effects of pregnancy or post-ovulatory progesterone (P(4)) supplementation on the expression of oestrogen and P(4) receptors (ESRs and PGRs) in the bovine uterus, heifers (n=263) were randomly assigned to the following treatments: i) cyclic, normal P(4); ii) cyclic, high P(4); iii) pregnant, normal P(4); and iv) pregnant, high P(4) on days 5, 7, 13 and 16 of pregnancy/oestrous cycle. Elevated P(4) was achieved through P(4)-releasing intravaginal device insertion on day 3 after oestrus, resulting in increased concentrations from day 3.5 to 8 (P<0.05) in the high groups than in the normal groups. Irrespective of treatment, PGR and ESR1 mRNA expressions were highest on days 5 and 7 and decreased on day 13 (P<0.05), while ESR2 mRNA expression increased on day 7 (P<0.05) and similar levels were maintained within the normal P(4) groups subsequently. Expression in the high P(4) groups decreased on day 13 (P<0.05). PGR-AB and PGR-B protein expressions were high in the luminal and superficial glands on days 5 and 7, but by day 13, expression had declined to very low or undetectable levels and high P(4) concentration tended to decrease or decreased significantly (P<0.05) the expression in these regions on days 5 and 7. ESR1 protein expression was high, with no treatment effect. ESR2 protein was also highly expressed, with no clear effect of treatment. In conclusion, early post-ovulatory P(4) supplementation advances the disappearance of PGR protein from the luminal epithelium on days 5 and 7, and decreases ESR2 mRNA expression during the mid-luteal phase, but has no effect on PGR or ESR1 mRNA expression.