State University of

Santa Cruz

Center for Research in

Reproduction and
Endocrinology

Rafaella Valete Nunes

Paiva
 The Uterine Melatonergic Systems
Impact of AANAT and Melatonin
Factor
Membrane Receptor 2 (MT2) are essential
5.6 for Endometrial
Receptivity and Early Implantation in Mice

2023
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 Pregnancy

I
n
t
r
o
d  Molecular and histological
u alterations [7,8].
c
 Coordinated by the steroid E2 and
t
P4 as well as other growth factors
i [6-8].
o
 Miscarriages, 75% of  Mucosal differentiation and
n
the cases [4]. immune cell [9-12].
 Prerequisite for successful
pregnancy [1].
 Meticulously timed [1].
Figure: WANG et al., 2006. 3
I Melatonin
n
t
r
o
d
u  Humans: Fertilization rates [41]
c
 Mouse: AANAT and MT2: blastocyst biological actions
t
i implantation and litter size [42] Membran
o e receptor
n  Mouse: Stimulates estradiol levels (E2) [43]
 Humans: Antioxidant enzymes [44]
 Animal model

Aanat knockout: ICR mice Mt2 knockout: C57BL/6J mice

M
e
t
h Mated
o
d
s Tissue Acquisition Techniques

Days of pregnancy (dpc): • Immunohistochemistry

⁻ 1.5 dpc • Western Blot
Uteri were collected ⁻ 2.5 dpc • Real-Time qPCR
⁻ 3.5 dpc • RNA-Seq Analysis
*Fetuses
⁻ 4.5 dpc
⁻ 13.5 dpc* 5
 Identification of Expression of AANAT, MT1, and MT2 in Uteri and Their KO for Mice Fertility

Figure 1

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(A–C) Aanat, Mt1, and Mt2 expression during pregnancy in mouse uteri. p < 0,05. 6
 Identification of Expession of AANAT, MT1, and MT2 in Uteri and Their KO for Mice Fertility

Figure 1 Figure 1

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(D,E) AANAT and MT2 immunohistochemistry. p < 0,05. 7

 Identification of Expression of AANAT, MT1, and MT2 in Uteri and Their KO for Mice Fertility

Figure 1

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(F–G) Fertility assessment in the control and Aanat KO, control and Mt2 KO mice. **p < 0,01. 8
 Effects of Aanat KO on Embryo Implantation and Uterine E2 and P4 as Well as Their Related
Downstream Gene Responses at 4.5 dpc
Figure 2

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(A,B) Implantation sites at 4.5 dpc, implantation window. (C) Immunohistochemical. **p < 0,01. 9
 Effects of Aanat KO on Embryo Implantation and Uterine E2 and P4 as Well as Their Related
Downstream Gene Responses at 4.5 dpc
Figure 2

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(D) Western blotting of BMP2 with statistical analysis. *p < 0.05; **p < 0.01. 10
 Effects of Aanat KO on Embryo Implantation and Uterine E2 and P4 as Well as Their Related
Downstream Gene Responses at 4.5 dpc
Figure 2

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(E) HE staining of 4.5 dpc uteri. (F) Western blotting of the FOXA2 LIF protein. *p < 0.05; **p < 0.01. 11
 Figure 2

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(G) Immunohistochemical ERα and PR. (H) Western blotting of the Erα. (I) Western blotting of the PR-A. *p < 0.05; **p < 0.01. 12
 Figure 2

Estrogen

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Progesterone

(J) Expression of the downstream response genes of estrogen and progesterone by PCR. *p < 0.05; **p < 0.01. 13
 Effects of Aanat KO on Global Gene Expression in Uteri during the Window of Implantation at
4.5 dpc in Mice
Figure 3

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(A) KEGG cluster analysis.

14
 Effects of Aanat KO on the ECM−Receptor Interaction at 4.5 dpc in Mice
Figure 3

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(B–D) Western blotting of ECM, CK8, MMP2, MMP9, MUC1, and CLDN1, respectively, with statistical analyses. * p < 0.05. 15
 Effects of Aanat KO on STAT Signaling at 4.5 dpc in Mice
Figure 3

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(E) Transcription factor differential analysis. (F) Western blotting of JAK–STAT with statistical analysis. * p < 0.05. 16
 Effects of Mt2 KO on Uterine E2 and P4 Responsiveness and Glands Formation at 4.5 dpc
Figure 4

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(A,B) Implantation sites during the implantation window. 17

 Effects of Mt2 KO on Uterine E2 and P4 Responsiveness and Glands Formation at 4.5 dpc
Figure 4

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(C) Expression of the BMP2 protein. (D) Expression of the ERα protein. (E) Expression of the PR-A protein. * p < 0.05. 18
 Effects of Mt2 KO on Uterine E2 and P4 Responsiveness and Glands Formation at 4.5 dpc
Figure 4

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(F,G) Quantitative PCR analysis of estrogen and progesterone response-related genes. * p < 0.05. 19
 Effects of Mt2 KO on Uterine E2 and P4 Responsiveness and Glands Formation at 4.5 dpc
Figure 4

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(H) HE and immunohistochemical staining of the uterus in 4.5 dpc mice. (I) Expression of FOXA2 . ** p < 0.01. 20
 Effects of Mt2 KO on Abortion and Uterine Immunity during the Second Trimester of Pregnancy
Figure 5

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(A–C) Uterine morphology. I–III shows different degrees of morphological abnormalities. * p < 0.05. 21
 Figure 5

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(D) GO enrichment analysis. (E–I) QPCR validation of GO term-related genes in uteri of 13.5 dpc. * p < 0.05. 22
 Figure 5

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(J) KEGG enrichment analysis. (K) QPCR validation of KEGG term-related genes. (L) Cytokines in uteri of 13.5 dpc* p < 0.05. 23
 • Placental maldevelopment and reduced early implantation sites in AANAT and MT2 KO.

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Figure 6. Schematic of the melatonergic systems (AANAT, MT2) that are active during embryo implantation. 24
 Thank
for your attention!
When you love the paper, but need to learn a lot!

Contact: rvnpaiva@uesc.br / +55 73 99187-7767

  Wang, H., Dey, S. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7, 185–199
(2006). https://doi.org/10.1038/nrg1808.

 Amaral FG do, Cipolla-Neto J. A brief review about melatonin, a pineal hormone. Arch Endocrinol Metab
[Internet]. 2018Jul;62(4):472–9. Available from: https://doi.org/10.20945/2359-3997000000066.
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