In-vitro construction of endometrial-like epithelium using CD146+ mesenchymal cells derived from human endometrium.

Endometrial CD146+ cells were purified, using magnetic activated cell sorting, and then embedded and cultured in a collagen-matrigel scaffold on top of myometrial smooth muscle cells for 10 days. At the end of culture period, the differentiation and formation of the epithelial-like cells were confirmed by morphological and ultrastructural evaluations, and analysis by reverse transcription polymerase chain reaction of the specific expression of genes: osteopontin (SPP1), matrix metalloproteinase 2, zonula occludens 1, laminin alpha 2 and collagen type IV; and by western blotting of CD9 protein. The results showed that the human endometrial mesenchymal CD146+ cells were able to produce endometrial glandular tube-like structures in vitro. Ultrastructural observation revealed some projections on the apical surfaces, appearance of basal lamina-like structures on the basal surface, and tight junctions and desmosomes on the lateral surfaces of the epithelial-like cells. The expression of studied genes at RNA level and CD9 at protein level confirmed the formation of endometrial epithelial-like cells. This culture system may have potential applications in cell therapy and in studies on human embryo implantation.

GnRH Neuron-Specific Ablation of Gαq/11 Results in Only Partial Inactivation of the Neuroendocrine-Reproductive Axis in Both Male and Female Mice: In Vivo Evidence for Kiss1r-Coupled Gαq/11-Independent GnRH Secretion.

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

The pregnant mouse uterus exhibits a functional kisspeptin/KISS1R signaling system on the day of embryo implantation.

BACKGROUND: Expression of kisspeptin (protein) and Kiss1r (mRNA) was recently documented in the mouse uterus on D4 of pregnancy (the day of embryo implantation) suggesting that the uterine-based kisspeptin (KP)/kisspeptin receptor (KISS1R) signaling system regulates embryo implantation. Despite this important suggestion, it was never demonstrated that the uterus actually exhibits a functional KP/KISS1R signaling system on D4 of pregnancy. Thus, the goal of this study was to determine whether a functional KP/KISS1R signaling system exists in the mouse uterus on D4 of pregnancy. FINDINGS: Since kisspeptin/KISS1R signaling triggers the phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, through immunohistochemical analyses, we determined whether exogenously administered kisspeptin could trigger p38 and ERK1/2 phosphorylation in the uterus on D4 of pregnancy. The results clearly demonstrated that kisspeptin could and that its effects were mediated via KISS1R. Additionally, the robust kisspeptin-triggered response was observed in the pregnant uterus only. Finally, it was demonstrated that on D4 of pregnancy the Kiss1 null uterus expresses functional KISS1R molecules capable of mediating the effects of kisspeptin. CONCLUSIONS: These results lead us to conclude that on D4 of pregnancy, the mouse uterus expresses a functional KP/KISS1R signaling system strengthening the possibility that this signaling system regulates embryo implantation. These findings strengthen the rationale for determining whether such a functional system exists in the uterus of the human female and if so, what role it might play in human pregnancy.

Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency.

BACKGROUND: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. OBJECTIVE: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. MATERIALS AND METHODS: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. RESULTS: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049). The rate of CD90 positive cells was significantly increased in LIF-treated group (98.96±0.37%) compared to control group (94.26±0.08%) (p=0.0498). Also, the expression ratio of all studied genes was significantly increased in the LIF-treated group compared to control group (p=0.0479). CONCLUSION: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

The effect of stem cell factor on proliferation of human endometrial CD146(+) cells.

BACKGROUND: Stem cell factor (SCF) is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. OBJECTIVE: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146(+) cells. MATERIALS AND METHODS: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS) into CD146(+) cells. Human endometrial CD146(+) cells were karyotyped and tested for the effect of SCF on proliferation of CD146(+) cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146(+) cells proliferation was assessed by MTT assay. RESULTS: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146(+) cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01). CONCLUSION: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146(+) cells and it has important implications for medical sciences and cell therapies.

Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect.

BACKGROUND: The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage. METHODS: Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105. RESULTS: 11.88 ± 1.29% of human endometrial cells whitin tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. CONCLUSION: The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.

Differentiation of human CD146-positive endometrial stem cells to adipogenic-, osteogenic-, neural progenitor-, and glial-like cells.

The aim of this study was to investigate the potential differentiation of CD146(+) endometrial stem cells to several lineages. Endometrial stromal cells were cultured using Dulbecco's modified Eagle's medium/Hams F-12 (DMEM/F-12) and were passaged every 7-10 d when cultures reached 80-100% of confluency. The immunophenotypes of single endometrial cells were analyzed using flow cytometry at fourth passage. Then the CD146(+) cells were sorted using magnetic-activated cell sorting, and they were cultured and analyzed for in vitro differentiation to several lineages. Detection of adipocyte- and osteocyte-like cells were assessed by oil red O and alizarin red staining, respectively. For detection of neural progenitor and oligodendrocyte-like cells, the cells were immunostained by neurofilament 68 and oligo2, respectively. The rates of CD90, CD105, CD146, CD31, CD34, and CD9 of cultured endometrial cells were 94.98 ± 3%, 95.77 ± 2.5%, 27.61 ± 2%, 0.79 ± 0.05%, 1.43 ± 0.1%, and 1.01 ± 0.06%, respectively. CD146(+) cells were isolated to high purity. CD146(+)-differentiated cells to adipogenic cell with typical lipid-rich vacuoles and osteogenic cells were observed and confirmed their mesenchymal origin. They also differentiated into neural progenitor and glial differentiation by retinoic acid, basic fibroblast growth factor, and epidermal growth factor signaling molecules, respectively, and confirmed by neurofilament 68 and oligo2 immunocytochemistry. The efficiency of differentiation to neural progenitor and oligodendrocyte-like cells was 90 ± 3.4% and 79 ± 2.8%, respectively. This study showed that CD146(+) cells from human endometrium after in vitro cultivation can differentiate into adipogenic-, osteogenic-, neural progenitor-, and glial-like cells. They may provide available alternative source of stem cells for future cell-based therapies and tissue engineering applications.

Ovarian stimulation by exogenous gonadotropin decreases the implantation rate and expression of mouse blastocysts integrins.

BACKGROUND: Integrins are heterodimeric glycoprotein receptors that regulate the interaction of cells with extracellular matrix and may have a critical role in implantation. The aim of this study was to investigate the effect of ovulation induction on the expression of α4, αv, ß1, and ß3 integrins in mouse blastocyst at the time of implantation. METHODS: The ovarian stimulated and non-stimulated pregnant mice were sacrificed on the morning of 5th day of pregnancy. The blastocysts were collected, and the expression of αv, α4, ß1, and ß3 integrins was examined using real-time RT-PCR and immunocytochemical techniques, then their ovarian hormones were analyzed at the same time. The implantation sites in uterine horns of other pregnant mice in both groups were determined under a stereomicroscope on the 7th day of pregnancy. RESULTS: The results showed that the expression of αv, ß1, and ß3 integrins in both mRNA and protein levels was significantly lower in the ovarian stimulated group than the control group, and the maximum ratio of expression was belonged to ß1 molecule (P>0.05). CONCLUSION: The implantation rate in superovulated mice was significantly lower than control mice. It was suggested that ovulation induction decreased the expression of αv, ß1, and ß3 integrins of mouse blastocysts.

Expression of α4, αv, ß1 and ß3 integrins during the implantation window on blastocyst of a mouse model of polycystic ovarian syndromes.

BACKGROUND: It has been hypothesized that blastocyst integrin expression changes can affect the spontaneous miscarriage in polycystic ovarian syndromes (PCOS). OBJECTIVE: In this study, the profile of integrin genes and proteins was investigated on blastocyst of the PCOS experimental mouse model. MATERIALS AND METHODS: 30 NMRI female mice were equally divided into 3 groups: control, experimental [PCOS that was injected estradiol valerate (40 mg/kg)]. After 8 weeks, each group was hyper stimulated by PMSG and HCG. Vaginal plaque was checked, and mice were investigated 5 days after the test. Progesterone and estradiol levels were determined; α4, αv, ß1 and ß3 integrin genes and protein of blastocysts were examined by real time PCR method and immunohistochemistry, respectively. RESULTS: Estradiol level was significantly increased (p≤0.035) in PCOS group. Based on our finding, the ratio of genes' expressions αv, ß3, ß1 and α4 in PCOS to control group was 0.479±0.01, 0.5±0.001, 2.7±0.4 and 1.023±0.2 respectively. Genes expression showed a great difference (p≤0.001) between ß3, ß1 and αv in PCOS compared to other groups. αv and ß3 integrin proteins expressed in all groups but intensity of these proteins in PCOS groups, was lower than other groups. CONCLUSION: Pattern of αv and ß3 integrins expression on the mouse blastocyst surface has an important effect during the implantation window. This pattern has changed in PCOS model and might have a great influence on implantation failure. Therefore, this experimental study suggests that a great attention to this problem may be essential in patients who are involved.

Assessment of α4, αv, ß1 and ß3 integrins expression throughout the implantation window phase in endometrium of a mouse model of polycystic ovarian syndromes.

BACKGROUND: Endometrial integrin expression changes might be a reason for implantation failure in polycystic ovarian syndromes (PCOS). Objective : Assessment of integrin genes and proteins expression upon endometrium in the PCOS experimental mouse model was the main goal of this study. MATERIALS AND METHODS: 30 NMRI female mice were equally divided into control, experimental (PCOS; received estradiol valerate (40 mg/kg)) and sham group (received; olive oil). After 8 weeks, each group was hyper stimulated by 7 IU PMSG and then, after 48hrs, 7 IU HCG was injected. Vaginal plaque was checked. After 5 days, Progesterone and estradiol levels and endometrial tissues were investigated to evaluate of α4, αv, ß1 and ß3 integrins gene and protein by qPCR method and immunohistochemistry, respectively. RESULTS: Tissue samples were assessed and showed that level of progesterone was significantly decreased in PCOS group. RESULTS of molecular part in the amount of αv, ß3, ß1 and α4 gene expressions showed a great difference in ß3 and αv genes expressions between experimental groups. αv, ß3, α4 and ß1 proteins in the endometrial stroma in the control group were expressed, but they were not detected in PCOS group. CONCLUSION: According to the results, integrins had different expression patterns in different areas of the endometrium; such as epithelial and stromal. It seems that in PCOS, this pattern has changed and the results might have a great influence on implantation failure. Therefore, this study suggests that a great attention to this problem may be essential in patients who are involved.