Metformin ameliorates endometrial thickness in a rat model of thin endometrium.

Metformin, a well-established anti-diabetic drug, is also used in managing various other metabolic disorders including polycystic ovarian syndrome (PCOS). There are evidences to show that metformin improves endometrial functions in PCOS women. However, fewer studies have explored the direct effects of metformin on endometrium. Previous in vitro studies have shown that therapeutic serum concentrations of metformin enhance endometrial epithelial cell proliferation. The present study was undertaken to investigate in vivo effects of metformin on endometrial proliferation in a rat model of thin endometrium. Toward this, a rat model of thin endometrium was developed. Metformin (0.1% or 1% w/v) was administrated orally for 15 days in rats with thin endometrium. Oral metformin administration for three consecutive estrous cycles (15 days) in the thin endometrium rat model led to an increase in endometrial thickness compared to sham endometrium. Histological analysis showed a significant increase in the number of endometrial glands (P < 0.05), stromal cells (P < 0.01) and blood vessels (P < 0.01) in metformin-treated (n = 10 in each group) uterine horns compared to sham (saline-treated) uterine horns in rats. The expression of proliferating cell nuclear antigen and vascular epithelial growth factor was found to be upregulated on treatment with 1% metformin-treated group (n = 7). However, pregnancy outcomes in the rats treated with metformin remained unaltered despite the restoration of endometrial thickness. In conclusion, the study demonstrated that metformin ameliorates endometrial thickness in a rat model of thin endometrium by increasing endometrial proliferation and angiogenesis, without restoration of embryo implantation.

Immunomodulatory Role of Surfactant Protein-D in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model.

Surfactant protein D (SP-D), a pattern recognition molecule, is emerging as a potent anti-tumoural innate immune defense molecule in a range of cancers. Previously, SP-D expression was found to be significantly downregulated at the malignant sites of human prostate adenocarcinoma and associated with an increasing Gleason score and severity. We recently reported selective induction of intrinsic apoptosis by a recombinant fragment of human SP-D (rfhSP-D) in the human Prostate cancer (PCa) biopsy explants and cells with glucose regulated protein of 78 (GRP78) as one of the key interacting partners. The present study evaluated the expression of SP-D in early and advanced stages of PCa using transgenic adenocarcinoma of mouse prostate (TRAMP) model. Both early and late stages of PCa showed significantly decreased SP-D mRNA expression and increased proteolytic degradation of SP-D protein. Systemic and tumoural immunophenotyping of TRAMP model revealed increased serine proteases producing granulocytes and polymorphonuclear myeloid-derived suppressor cells (PMN MDSCs) in the late stage; the serine proteases secreted by these cells could be involved in the degradation of SP-D. Susceptibility of rfhSP-D to elastase-mediated proteolysis provided the rationale to use an elastase-inhibitor to sustain intact rfhSP-D in the tumour microenvironment. The study revealed an immunomodulatory potential of rfhSP-D and elastase inhibitor, sivelestat, to induce macrophage polarization towards M1 with downregulation of PMN MDSCs in ex-vivo cultured TRAMP tumours. Furthermore, rfhSP-D induced immunogenic cell death in murine PCa cells and in TRAMP explants. The findings highlight that SP-D plays an anti-tumourigenic role in PCa by inducing immunogenic cell death and immunomodulation while the prostate tumour milieu adversely impacts SP-D by inhibiting its transcription, and enhancing its proteolytic degradation. Transformation of an immunologically "cold tumour" into a "hot tumour" implicates therapeutic potential of rfhSP-D in PCa.

Additional evidence to support OCT-4 positive VSELs and EnSCs as the elusive tissue-resident stem/progenitor cells in adult mice uterus.

OBJECTIVE: True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS: This was achieved by undertaking three sets of experiments. Firstly, SSEA-1+ and Oct-4 + positive VSELs, sorted from GFP mice, were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly, uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly, OCT-4 expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS: GFP + ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident, nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs, cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION: Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also, VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies.

Mice Uterine Stem Cells are Affected by Neonatal Endocrine Disruption & Initiate Uteropathies in Adult Life Independent of Circulatory Ovarian Hormones.

It is generally believed that ovarian hormones regulate uterine functions and their altered levels result in various uteropathies like non-receptive uterus, endometrial hyperplasia, adenomyosis, endometriosis, leiomyomas and cancer. Uterus harbors two populations of stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-specific progenitors (endometrial stem cells, EnSCs). Unlike endometrial mesenchymal stem/ stromal cells, VSELs/EnSCs express ERα, ERß and PR which makes them directly vulnerable to perinatal endocrine insults. Present study was undertaken to evaluate whether uteropathies occur due to altered hormones and/or intrinsic changes in stem/progenitor cells. Mice pups, exposed to estradiol (20 µg/pup/day) on postnatal days 3-7 or vehicle, were subjected to bilateral ovariectomy on day 30 and later exposed sequentially to estradiol and progesterone resulting in receptive uterus in control mice. Despite similar hormonal exposure, endocrine disruption resulted in non-receptive uterus with noticeable endometrial and myometrial hyperplasia and up-regulation of stem cell markers (Oct-4A, Oct-4, Sox2, Nanog). Glands were poorly formed and 'defective' epithelial progenitors were found disseminated into myometrium and blood vessels revealing how adenomyosis and endometriosis possibly initiate. Progesterone resistance and estradiol dominance due to downregulation of Erα & Pr and upregulation of Erß transcripts was observed in both intact uterus and stem cells enriched from uterus. Transcripts specific for DNA mismatch repair axis (Pcna, NP95 and Dnmt1), repair enzymes (Brca-1, Rad51 and Mlh1) were dysregulated whereas Ki67 was ten-folds increased suggestive of genomic instability. Study reveals role of stem cells in initiating uteropathies during adult life independent of circulatory ovarian hormones. Endocrine disruption affects tissue resident stem/progenitor cells (VSELs/EnSCs) in both endometrium and myometrium, result in epithelial cells hyperplasia, non-receptive endometrium, adenomyosis and defective stem cells and epithelial progenitors were detected in the perimetrium from where they can mobilize to ectopic sites to initiate endometriosis. Study shows stem cell basis for various uteropathies. VSEL: Very small embryonic like stem cell; EnSC: Endometrial stem cell; E + P: Estradiol + Progesterone; E: Endometrium; P: Perimetrium; M: Myometrium; ACD: Asymmetrical cell division; SCD: Symmetrical cell division; CE: Clonal expansion; G: Gland; S: Stromal cell; US: Undifferentiated stromal cell; LE: Luminal epithelium; GE: Glandular epithelium; EP: Epithelial progenitors; SMC: Spindle-shaped myometrial cell; OMC: Oval-shaped myometrial cell.

Effect of Aging and 5-Fluorouracil Treatment on Bone Marrow Stem Cell Dynamics.

Lifelong homeostasis of bone marrow is maintained by the resident stem cells that include the quiescent very small embryonic-like stem cells (VSELs) and lineage restricted, tissue committed 'progenitors' hematopoietic stem cells (HSCs). Niche providing mesenchymal stromal cells (MSCs) regulate the function of VSELs/HSCs by providing crucial paracrine support. Any dysfunction of stem cells and/or their niche leads to disease state. The stem cells biology gets affected with age leading to a myeloid bias in differentiation of HSCs and increased incidence of myeloid leukemia. Present study was undertaken to enumerate VSELs, HSCs and MSCs and evaluate their response on D4 and D10 after chemotherapy with 5-Fluorouracil (5-FU) in young and aged mouse bone marrow. Stem cells were activated in response to 5-FU induced stress in an attempt to restore homeostasis. Although absolute numbers of VSELs and HSCs did not differ much between young and aged mice, their tendency to proliferate was higher on D4 in aged mice. However, ability to revert back to basal numbers and their differentiation was affected on D10 in aged marrow. Stem cells from aged bone marrow showed greater ability to form CFUs on D10 after 5-FU treatment. CD44 positive aged MSCs also showed increased proliferation on D10. Transplanting MSCs from young mice in 5-FU treated aged marrow helped improve hematopoiesis. The results suggest that no significant intrinsic changes occur as proliferative ability of stem cells remains unaffected but the niche gets affected with age leading to excessive self-renewal and compromised differentiation. This may explain increased incidence of leukemia with age.

Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy.

Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6 µm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 µm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.

Use of ultrasound imaging for the diagnosis of abnormal uterine bleeding in the bonnet macaque ( Macaca radiata).

Ultrasound is a powerful, low-cost, non-invasive medical tool used by laboratory animal veterinarians for diagnostic imaging. Sonohysterography and transvaginal ultrasound are frequently used to assess uterine anomalies in women presenting with abnormal uterine bleeding (AUB). In the present study, we have evaluated the abdominal ultrasound of bonnet monkeys ( n = 8) showing spontaneous ovulatory ( n = 5) and anovulatory ( n = 3) AUB. The ovulatory ( n = 5) macaques showed cyclic AUB for 7-8 days. The anovulatory ( n = 3) macaques had irregular AUB with menstrual cycles of 40-45 days. The B-mode abdominal, colour Doppler and 3D ultrasound scans were performed during the proliferative phase of the menstrual cycle. Ultrasound examination revealed endometrial polyps in five macaques and endometrial hyperplasia in three animals. The width and length of endometrial polyps was around 0.5-1 cm (average 0.51 ± 0.23 cm × 0.96 ± 0.16 cm) with significant increase in endometrial thickness ( P < 0.0002). 3D ultrasound also showed a homogeneous mass in the uterine cavity and colour Doppler ultrasound showed increased vascularity in the endometrial polyps. Endometrial hyperplasia characteristically appeared as a thickened echogenic endometrium ( P < 0.0002). This study demonstrates the use of non-invasive ultrasound techniques in the diagnosis of AUB in macaques.

Very small embryonic-like stem cells are the elusive mouse endometrial stem cells--a pilot study.

BACKGROUND: Endometrium undergoes dramatic growth, breakdown and regeneration throughout reproductive period in mammals. Stem cells have been implicated in the process however their origin, nature, anatomical localization and characterization still remain obscure. Classical concept of presence of stem cells in the basal layer of endometrium was recently challenged when side population and label retaining cells were found to be distributed throughout endometrium. We have earlier reported very small embryonic-like stem cells (VSELs) in adult mammalian ovary and testis as a small population of cells with nuclear OCT-4 along with progenitors (spermatogonial stem cells and ovarian germ stem cells) with cytoplasmic OCT-4. Present study was undertaken to gauge presence of VSELs in bilaterally ovariectomized mouse uterus and their modulation by hormones. METHODS: Bilaterally ovariectomized mice were subjected to sequential estradiol and progesterone treatment in order to induce proliferation, differentiation and remodeling (regeneration). Stem cells were studied in tissue smears after H & E staining and after sorting using SCA-1 by immuno-localization and qRT-PCR studies (Oct-4A, Nanog and Sca-1). Flow cytometry studies were also undertaken to confirm the presence of VSELs in mouse uterus. RESULTS: Two distinct populations of stem cells with dark stained nucleus and high nucleo-cytoplasmic ratio were detected in ovariectomized mouse uterus. These cells were sorted using SCA-1 and comprised smaller VSELs with nuclear expression of OCT-4 and slightly bigger, more abundant progenitors termed as endometrial stem cells (EnSCs) with cytoplasmic OCT-4. RT-PCR studies showed presence of pluripotent transcripts (Oct-4, Sca-1) and flow cytometry confirmed the presence of 0.069% of LIN-/CD45-/SCA-1+ VSELs. These stem cells were distinctly regulated during endometrial growth, differentiation and regeneration as evidenced by qRT-PCR results. CONCLUSIONS: VSELs are present in normal uterus and also under conditions of atrophy induced by bilateral ovariectomy. Marked increase in EnSCs is associated with endometrial growth and regeneration. Further studies are warranted to define the niche for these stem cells and whether EnSCs arising from the pluripotent VSELs are common progenitors for epithelial and stromal cells or not remains to be addressed. Results of the present study will help in better understanding of endometrial pathologies and their management in the future.

Ovarian Hormones Regulate SP-D Expression in the Mouse Uterus During Estrous Cycle and Early Pregnancy.

PROBLEM: Differential expression of SP-D in the cycling human and mouse endometrium suggests its regulation by ovarian hormones. METHOD OF STUDY: SP-D expression in the mouse uterus was analyzed across the estrous cycle and during early pregnancy. Effect of exogenous ovarian hormones on the uterine expression of SP-D was analyzed. RESULTS: SP-D expression varied across the estrous cycle and peaked in the estrous phase. SP-D transcript levels increased by fourfold in the uteri of estrogen-treated mice while co-administration of estrogen and progesterone enhanced SP-D levels by ninefold. However, treatment with progesterone alone significantly downregulated SP-D expression. Diethylstilbestrol enhanced SP-D transcript levels in the uteri of immature mice by 10-fold. During pregnancy, SP-D levels declined rapidly from 0.5 dpc to 6.5 dpc. In silico analysis predicted the presence of two potential ERE and 1 PRE in the mouse SP-D gene promoter region. CONCLUSION: Estrogen positively regulates expression of SP-D in the mouse uterus. Progesterone, along with estrogen synergizes SP-D expression, however, when administered alone results in negative regulation.

Proliferation and decidualization of endometrial stromal cells during embryo-attachment stage in bonnet monkeys (Macaca radiata).

We report embryo-induced alterations occurring in endometrial stromal cells (ESCs) during the embryo-attachment stage in bonnet monkeys (Macaca radiata). Laser micro-dissected ESCs obtained from pregnant and non-pregnant animals were compared for levels of selected proliferation and decidualization-associated factors by analysis with quantitative real-time polymerase chain reaction or immunohistochemistry. Stromal cells exhibited extensive cellular proliferation, as indicated by cellular compaction and significantly higher (P < 0.05) levels of proliferating cell nuclear antigen and of estrogen receptor 1, c-Myc, and Cyclin D1 transcripts in pregnant animals as compared with non-pregnant animals. A significant decrease (P < 0.05) was observed in the transcript levels of stromal interleukin-6 (IL-6) in pregnant animals. Cell proliferation was accompanied by a significant increase (P < 0.001) in the levels of decidualization-associated molecules such as IL-1ß in the luminal and glandular epithelium and of stromal insulin-like growth-factor-binding protein-1 (IGFBP-1) and prostaglandin-endoperoxide synthase-2 (PTGS-2) proteins. In pregnant animals, proliferation was evident throughout the gestational stroma, whereas decidualization was more pronounced in the embryo-attachment zone than in the non-attachment zone. To our knowledge, this is the first report of alterations in the endometrial stroma during the embryo-attachment stage in a non-human primate model.