Dhananjay D. Manjramkar

Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture

BackgroundCryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells.MethodsSerum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR.ResultsA prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition.ConclusionThe present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Very Small Embryonic-Like Stem Cells Survive and Restore Spermatogenesis after Busulphan Treatment in Mouse Testis

Study Objectives: Adult mammalian testes harbor a novel population of quiescent, pluripotent, very small embryonic-like stem cells (VSELs) along with spermatogonial stem cells (SSCs). Present study was undertaken to (i) characterize testicular VSELs (ii) investigate differential effect of chemotherapy on VSELs and SSCs and (iii) to restore the differentiation ability of surviving VSELs by providing healthy somatic microenvironment. Methods: Effect of busulphan (25 mg/Kg) was studied on mouse testes. Syngenic Sertoli cells(105 cells per testis) and bone marrow derived mesenchymal cells (104 cells per testis) were transplanted separately through intertubular route. Effect of niche reconstruction was studied two months later by histology and immuno-localization of germ cell markers MVH and PCNA. Caudal sperm were evaluated for their ability to fertilize oocytes in vitro. Results: VSELs were 2-6 μm in size, SCA-1+/CD45-/LIN-, had high nucleo-cytoplasmic ratio and comprised 0.03% of testicular cells whereas SSC specific marker GFRa localized on a distinct, larger cell population. Busulphan selectively destroyed SSCs and other germ cells however, nuclear OCT-4A, Nanog, Sox-2 and SCA-1 positive VSELs (0.06%) survived. Persisting VSELs were unable to differentiate possibly because chemotherapy also affected the ‘niche’ comprising Sertoli cells. Complete restoration of spermatogenesis was observed two months post transplantation of Sertoli and mesenchymal cells. Transplanted cells formed neo-tubules in the vicinity of surviving tubules and were possibly a source of growth factors essential for VSELs proliferation and differentiation. Both MVH and PCNA showed increased staining in the transplanted group. qRT-PCR studies revealed existence of a meiotic block in busulphan treated testis which was overcome after transplantation. Resulting sperm progressed to epididymis, showed normal motility and ability to fertilize in vitro. Conclusion: Results show that VSELs survive chemotherapy and can restore spermatogenesis in germ cells depleted mice. Results have direct relevance to address fertility issues of cancer survivors.

Expression Profiles of Endometrial Leukemia Inhibitory Factor, Transforming Growth Factor β2 (TGFβ2), and TGFβ2 Receptor in Infertile Bonnet Monkeys1

Abstract The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor β2 (TGFβ2), and transforming growth factor β2 receptor (TGFβ2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFβ2, and TGFβ2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFβ2, and TGFβ2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFβ2 and TGFβ2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFβ2 and TGFβ2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFβ2, and TGFβ2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFβ2, and TGFβ2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFβ2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to endometrial receptivity, and that any aberration in this step may adversely affect the subsequent molecular events (i.e., expression of LIF). These data also suggest that potential aberrations in the functional network of locally produced cytokines and growth factors even may occur in an endometrium exposed to the optimal peripheral hormonal levels.

Evaluation of the antifertility effect of magainin-A in rabbits: in vitro and in vivo studies

OBJECTIVE To evaluate the safety and contraceptive potential of magainin-A in rabbits. DESIGN Controlled laboratory study. SETTING Department of Immunology, Institute for Research in Reproduction, Mumbai, Parel, India. ANIMAL(S) Forty-eight female New Zealand white rabbits. INTERVENTION(S) The effect of magainin-A on sperm motility (in vitro and in vivo studies) and on vaginal epithelium (histologic study) was assessed along with serum and liver biochemical profiles. MAIN OUTCOME MEASURE(S) Suitability of magainin-A for contraceptive use. RESULT(S) Magainin-A arrested sperm motility, and 1 mg of magainin-A administered intravaginally blocked conception. No histopathologic abnormalities in the vaginal tissue or any changes in serum biochemical profiles were observed. CONCLUSION(S) Magainin-A may be used as an effective and safe intravaginal contraceptive compound that also has antibacterial properties.

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

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

BackgroundEndometrium 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.MethodsBilaterally 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.ResultsTwo 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.ConclusionsVSELs 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.

Endometrial modifications during early pregnancy in bonnet monkeys (Macaca radiata)

The present study was undertaken to investigate endometrial modifications that occur before embryo invasion in bonnet monkeys (Macaca radiata). These changes were analysed in luminal epithelium, glandular epithelium and stroma of endometrial functionalis on Day 6 post ovulation from pregnant and non-pregnant animals (n = 4 each) by transmission electron microscopy. Distinct features (i.e. loss of columnar shape by epithelial cells, changes in mitochondrial size and diffused apicolateral gap junctions) were observed in the luminal and glandular epithelium in pregnant animals. Stromal compaction was also observed in pregnant animals. Further, immunogold localisation studies demonstrated significantly higher expression (P < 0.05) of oestrogen receptor alpha, an oestrogen-regulated gene, in the glandular epithelium and stroma of the endometrium in pregnant animals compared with non-pregnant animals. Expression of two other genes known to be regulated by oestradiol, namely beta-actin and cyclo-oxygenase-1, were also significantly higher (P < 0.05) in the endometria of pregnant animals. These studies demonstrate marked changes in the endometrium before embryo invasion in bonnet monkeys. These studies also indicate altered oestrogenic activity in the uterine milieu before embryo invasion.

Embryo-induced alterations in the molecular phenotype of primate endometrium.

Reproductive biomedicine has made significant advances in the area of assisted reproductive technologies in the last two and half decades. However, embryo implantation remains a major obstacle in securing high pregnancy rates. Various non-human primate models including rhesus, marmoset and baboon have been employed to elucidate in vivo mechanisms underlying the uterine events that initiate, sustain and complete implantation. This review collates the information available on the molecular profile of gestational endometrium in primates. Collectively, these studies reveal dynamic spatio-temporal changes in the expression of cytokines, growth factors, cell-adhesion molecules, cytoskeleton elements and other factors in the endometrium during the post-implantation phase of pregnancy. Considering that the endometrial events during the pre-implantation stages of pregnancy may dictate implantation success, we have developed a bonnet monkey (Macaca radiata) model where pregnancy can be detected at the pre-implantation stage. Using this model, we investigated some of the endometrial events that occur before the completion of implantation. Remarkable changes were observed in endometrial expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha), as well as expression of immunosuppressive factors such as transforming growth factor beta-2 (TGFbeta2), interleukin-6 (IL-6) and placental protein-14 (PP-14), even before the embryo starts invading the endometrium. This highlights the super-imposition of endometrial receptivity by embryonic stimuli, marked by differential expression and/or localization of the factors that regulate endometrial transformation for embryo survival, growth and development.

Differential expression of calreticulin, a reticuloplasmin in primate endometrium

BACKGROUND To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). METHODS Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR. RESULTS Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P < 0.01) in glandular epithelium and (ii) PDI in stroma (P < 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P < 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P < 0.05 versus control), with a trend towards down-regulation by progesterone. CONCLUSION Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium.

Expression of Endometrial Protein Kinase A During Early Pregnancy in Bonnet Monkeys (Macaca radiata)

Embryo-induced signaling pathways are considered to be important for initiation and sustenance of pregnancy. However many of these pathways remain to be deciphered in primates. In the present study, differential display RT-PCR was used to identify genes or gene fragments that are differentially expressed in endometrium of bonnet monkeys (Macaca radiata) on Day 6 of pregnancy. Of several fragments found to be differentially expressed, a fragment of 567 base pair (named GG1) was characterized in detail. GG1 was highly represented in endometrium of pregnant animals compared with that of nonpregnant animals. Sequencing analysis revealed homology of this fragment to exons 7, 8, 9, and 10 and surprisingly to intron 6 of cAMP-dependent protein kinase A (PKA) regulatory type I alpha (tissue-specific extinguisher 1) (PRKAR1A). The increased expression of this fragment in gestational endometrium was confirmed by quantitative PCR studies. Two transcripts of 3.0 kilobase (kb) and 1.5 kb were detected in Northern blot probed with labeled GG1. Protein expressions of alpha regulatory (PRKAR1A) and alpha catalytic (PRKCA) subunits of PKA were also higher in gestational endometrium compared with that in nongestational endometrium. Further in vitro studies using human endometrial explants demonstrated regulation of PRKAR1A (or GG1) and prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2 (PTGS2) by estradiol. This is the first study to date on the differential expression of PKA in primate endometrium during early pregnancy and its in vitro regulation by estradiol.