Coordination of both embryo and uterus is required at the ‘window of implantation (WOI)’ or ‘window of receptivity (WOR)’ for successful embryo implantation [27]. At this particular time, the blastocyst can survive and implant in the uterine milieu, which is otherwise hostile. The collective action of two key ovarian hormones, estrogen and progesterone, is a prerequisite for embryo implantation. These hormones bind to their corresponding nuclear receptor and act on their downstream targets [28]. Most of the genes involved in embryo implantation are responsive to the two ovarian steroids. However, recently some genes have been reported which are not responsive to estrogen, progesterone, or both. Previous studies reported that Kruppel-like factor 5 (Klf5), a zinc-finger-containing transcription factor, is unresponsive to ovarian steroids in the uterus [15]. In the present investigation, we studied the expression pattern of Klf5 during the periimplantation period. The effect of antiestrogen (ICI 182,780) and antiprogesterone (RU486) on the expression pattern of Klf5 has also been studied in the present study.
Embryo implantation is the process of a tightly regulated sequence of events. The blastocysts remain floated in the uterine lumen for a certain period before implantation [29]. D4.5 of gestation is the crucial time for uterine receptivity. During this time, the plasma membrane of uterine luminal epithelium undergoes drastic changes to make non-receptive uterus to the receptive state through an event known as plasma membrane transformation [30]. Under the influence of ovarian steroids, the uterine lumen changes to a slit-like structure which contributes to the apposition of blastocyst trophectoderm to the uterine luminal epithelium. The luminal epithelium is the first layer that comes in contact with implanting blastocyst. The KLF5 protein is localized in the luminal and glandular epithelium by Immunofluorescence study on D4 of gestation. From the results obtained, it can be speculated that this factor may have some role in epithelial cell differentiation and glandular secretion, which are prerequisites for implantation initiation. Localization of KLF5 in proliferating stromal cells indicates its role in embryo maintenance.
With the initiation of implantation, the embryos in the blastocyst stage are encased in a crypt (implantation chamber) formed by evaginations of luminal epithelium towards the antimesometrial pole for proper homing of the embryo. The ICM of the embryo is facing towards the mesometrial pole surrounded by polar trophectoderm. During this period, the transcription factor is localized in various embryo and uterus cell types. The stromal cells adjacent to implanting blastocyst undergo decidualization to form the primary decidual zone (PDZ) on D5 of gestation. Localization of KLF5 protein in the PDZ on D5 of gestation indicates its role in PDZ formation. PDZ acts as a barrier restricting the migration of immune cells or any undesired matters from the maternal circulation to the embryo. Results of our present study showed localization of KLF5 in the luminal epithelium and glandular epithelium suggests its role in embryo attachment and glandular secretion. With the progression of gestation, the lateral luminal epithelium surrounding the blastocyst undergoes apoptosis or entosis by the trophectodermal cell to accommodate the embryo in the stromal compartment [6, 31]. In conditionally ablated Klf5 mice uterus, the luminal epithelium remains intact at this particular time leading to implantation failure [15]. This suggests the role of Klf5 in luminal epithelial disintegration and successful embryo implantation.
On D6 of gestation, the embryo has invaded into the antimesometrial stromal layer, which is undergoing decidualization to form PDZ. The stromal cells next to PDZ start to undergo decidualization forming the secondary decidual zone (SDZ). The decidua provides nutrition and protection to the growing embryo. Our results showed the expression of the KLF5 transcription factor in the embryo and uterine compartment. The results obtained on D6 of gestation suggest that Klf5 has a role in the development of the embryo. Expression of this protein observed in the PDZ and SDZ of the uterine compartment suggests its critical role in the decidualization process, embryo invasion in maternal tissues, and stromal epithelial cross-talk during implantation.
The PDZ undergoes apoptosis to accommodate the growing embryo with the advancement of pregnancy. To meet the high energetic demand of decidualization, autophagy is important [32]. The KLF5 protein is localized in the proliferating stromal cells of PDZ and SDZ, ascertaining the role of this transcription factor in programmed decidualization and autophagy. On D7 of gestation, the egg cylinder becomes elongated. The expression of growth factor has been observed in the ectoplacental cone (EPC), distal endoderm, mural trophectoderm, primitive streak, and embryonic ectoderm on the D7 embryo. The developmental role of this transcription can be ascertained by looking at the results obtained.
On D8 of gestation, there is continuous disintegration and degradation of PDZ to make room for the developing embryo. The proliferating stromal cells of PDZ and SDZ show the localization of this transcription factor. These results consistently support the role of KLF5 in decidualization throughout the periimplantation period. The differential expression of KLF5 in various embryo cell types suggests the role of KLF5 in organogenesis.
To know the effect of estrogen and progesterone receptor antagonists, pregnant mice have been treated with the ovarian steroid antagonist. ICI 182,780 is an estrogen receptor antagonist which binds to both ERα and ERβ. The antiestrogen binds to the estrogen receptors (ER) and leads to reduced dimerization of the receptor, causing rapid receptor degradation [33]. Nucleocytoplasmic shuttling of ER-ICI 182,780 complex is disrupted [34]. Thus administration of ICI 182,780 blocks the activity of downstream targets of E2-ER signalling. In our present study, uterine luminal closure was not observed in the antiestrogen-treated mice uterus on D4 of gestation. The phenomenon of uterine luminal closure with the beginning of pregnancy has been considered one of the critical, required events of successful gestation. Antiestrogen-treated mice uterus showed intense immunostaining of the transcription factor KLF5 in the luminal epithelium. A previous study showed that downregulation of KLF5 in the luminal epithelium is needed to make the uterus receptive [15]. Intense immunostaining of KLF5 in luminal epithelium indicated its non-receptivity. It can be inferred from the present investigation that KLF5 uses other pathways for its functioning. The expression of this transcription factor Klf5 could be estrogen-ER signalling independent during periimplantation in mice uterus.
Progesterone receptor antagonist RU486 binds to PR and leads to a conformational change in PR, making the receptor inactive. RU486 blocks the progesterone functions [35, 36]. Antiprogesterone, RU486, decreases the height of microvilli and pinopodes in the apical surface of the plasma membrane [36, 37]. RU486 inhibits the proliferation of stromal cells as the basic fibroblast growth factor expression in stromal cells is inhibited, leading to compromised decidualization [38, 39]. Since RU486 blocks progesterone signalling, extensive epithelial proliferation was observed. Compromised luminal closure and reduced stromal edema were observed in the RU486-treated mice uterus when compared with D4. Intense expression of the transcription factor was observed in the luminal epithelium. From the findings of the present investigation, it has been speculated that KLF5 is not dependent upon the progesterone-PR pathway and operates in a different signalling cascade.
A similar expression pattern of the transcription factor was observed in the ICI 182,780+RU486-treated group. Co-treatment of antiestrogen and antiprogesterone blocks both estrogen and progesterone signalling cascade in mice uterus. Estrogen-induced epithelial proliferation and progesterone-induced stromal cell proliferation has been blocked in the treated mice uterus leading to aberrant luminal closure. As KLF5 was localized in the luminal and glandular epithelium in the antagonist-treated mice, it is believed that the expression of this transcription factor is not dependent upon estrogen or progesterone signalling.
Western blot analysis in the present investigation showed the presence of this protein in D4–D8 of gestation. On D4 of gestation, the least KLF5 protein level was reported, whereas the highest protein level was reported in D8 of gestation. There was a gradual increase in the protein level of the transcription factor with the progression of gestation. From the results obtained, it has been deciphered that KLF5 has a crucial role in uterine receptivity, implantation, and decidualization. In the ovarian steroid antagonist-treated sample, the presence of KLF5 protein was observed. The result showed that the expression of KLF5 is not dependent upon ovarian steroids, estrogen and progesterone. We can say from our findings that KLF5 has a paramount role in the implantation and maintenance of pregnancy.
Our real-time PCR result showed Klf5 mRNA transcript was upregulated with the progression of gestation from D4–D8. The presence of Klf5 mRNA transcript in ovarian steroid antagonist-treated mice uterus on D4 of gestation points out that the expression of this transcription factor is not dependent on key ovarian steroids, estrogen and progesterone.