Effect of 4-octylphenol on germ cell number in cultured human fetal gonads

This study evaluates whether a hormone disruptor found in environment, 4-octylphenol, affects the rate of proliferation of germ cells from human fetal gonads during a 3 week culture period. Five testis and five ovaries were obtained from fetuses of women undergoing legal abortions between the 6th and 9th week of fetal life, representing the period where early gonadal differentiation takes place. Each gonad was divided into equal sized test and control tissue. The test tissue was exposed to a continued presence of 10 micromol/l 4-octylphenol in the culture medium. The cultures were terminated by fixation of the tissues, which where then processed for histology and serially sectioned. The mitotic index of the germ cells (i.e. number of mitosis per 100 germ cells) and the number of germ cells per area was determined. Each of the five testes cultured in 4-octylphenol exhibited a significantly reduced mitotic index and number of pre-spermatogonia compared to the control, whereas none of the five ovaries exposed to 4-octylphenol revealed any difference compared to the control. It is concluded that 4-octylphenol exerts a sex-specific effect on male germ cells.     

Number of germ cells and somatic cells in human fetal ovaries during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal ovaries during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the gonads. METHODS: One ovary with attached mesonephros from each of 11 individual legal abortions was used for estimation of cell numbers. After recovery of the fetus, the ovary-mesonephric complexes were immediately isolated, fixed and processed for histology. A stereological method was utilized to estimate the total number of oogonia in all ovaries and somatic cells in seven of them. RESULTS: The number of oogonia per ovary increased from approximately 26,000 in week 6 to approximately 250,000 in week 9 and somatic cells from approximately 240,000 to approximately 1.4 x 10(6). The ratio of oogonia to somatic cells tended to increase throughout the period. The concentration of oogonia was similar in the cranial (mesonephric connected) part and the caudal part of the ovaries. CONCLUSIONS: This is the first stereological estimation of the number of oogonia and somatic cells in human fetal ovaries, and the first estimation of germ cells and somatic cells in ovaries aged <9 weeks. The number of oogonia in week 9 is comparable to the numbers previously published based on non-stereological estimations. We found early stages of meiosis in fetal ovaries from week 9.     

Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal testes during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the testes. METHODS: One testis with attached mesonephros from each of 10 individual legal abortions was used. After recovery of the fetus, the testes were immediately isolated, fixed and processed for histology. The optical fractionator technique, a stereological method, was utilized to estimate the total number of germ cells in ten testes and somatic cells in six of them. RESULTS: The number of germ cells per testis increased from approximately 3000 in week 6 to approximately 30000 in week 9. The ratio of germ cells to Sertoli cells was approximately 1:11 and the ratio of germ cells to somatic cells was approximately 1:44 throughout this period. CONCLUSIONS: For the first time, germ cell and somatic cell number have been determined during early human fetal testis development. Knowledge of the number of germ cells in this period may be very important, because several environmental pollutants are suspected to result in decreased semen quality in men born of mothers exposed to these pollutants during pregnancy.     

Expression of pluripotent stem cell markers in the human fetal ovary

BACKGROUND: Human primordial germ cells (PGCs) can give rise to pluripotentstem cells such as embryonal carcinoma cells (ECCs) and embryonicgerm cells (EGCs). METHODS: In order to determine whether PGCs express markers associatedwith pluripotency in EGCs and ECCs, the following study crossexamines the expression patterns of multiple pluripotent markersin the human fetal ovary, 5.5–15 weeks post-fertilizaton(pF) and relates this expression with the ability to derivepluripotent EGCs in vitro. RESULTS: Specific subpopulations were identified which included OCT4⁺/Nanog⁺/cKIT⁺/VASA⁺PGCs and oogonia. Interestingly, these cells also expressedSSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurredthroughout the entire gonad. Isolation of SSEA1⁺ cells fromthe gonad resulted in AP⁺ EGC colony formation. The number ofOCT4⁺ or Nanog⁺ expressing cells peaked by week 8 and then diminishedafter week 9 pF, as oogonia enter meiosis. In addition, theefficiency of EGC derivation was associated with the numberof OCT4⁺ cells. TRA-1-60 and TRA-1-81 were only detected inthe lining of the mesonephric ducts and occasionally in thegonad. CONCLUSIONS: These results demonstrate that PGCs, a unipotent cell, expressmost, but not all, of the markers associated with pluripotentcells in the human fetal ovary.     

Environmental toxicant-induced germ cell apoptosis in the human fetal testis

BACKGROUND: Disorders of the male reproductive system are increasing in prevalence. The term testicular dysgenesis syndrome emphasizes the importance of developmental influences on the aetiology of conditions including cryptorchidism, testicular germ cell cancer and reduced spermatogenesis. Men whose mothers smoked during pregnancy have lower sperm production. Cigarette smoke contains agents acting on the aryl hydrocarbon receptor (AHR). We have investigated the presence of AHR in the developing human testis and the effects of functional activation. METHODS AND RESULTS: Immunohistochemistry determined AHR to be expressed by germ cells in the human testis between 7 and 19 week gestation, but not by other cells. Treatment of cultured fetal testis with an AHR ligand present in tobacco smoke increased markers of cell apoptosis, and this was prevented by an AHR receptor antagonist. Immunohistochemistry indicated that apoptosis was restricted to germ cells. CONCLUSIONS: Germ cells in the developing human testis are a target for regulation by AHR ligands. Activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth, and may also be of importance in other conditions comprising the testicular dysgenesis syndrome.     

Oocyte donation in patients with Turner's syndrome: a successful technique but with an accompanying high risk of hypertensive disorders during pregnancy

BACKGROUND: Few data are available on pregnancy rate and obstetrical outcome after oocyte donation in Turner's syndrome patients. We conducted a retrospective analysis on the outcome of this subgroup. METHODS: Thirty oocyte donation cycles with fresh embryo transfer were performed in 21 patients between 2001 and 2004. RESULTS: The mean (+/-SD) age of the recipients was 33.1+/-1.8 years. The median (range) number of transferred embryos per cycle was two (1-4). Seventeen pregnancies were obtained (57%), of which 12 were clinical (40%). The implantation rate and the ongoing pregnancy rate were 22% (15 out of 68) and 30% (nine out of 30), respectively. Premature delivery was observed in 50% (four out of eight) of the pregnancies and intrauterine growth retardation in 55.5% (five out of nine) of the fetuses. Hypertensive disorders occurred in five out of eight pregnancies (three pre-eclampsias). CONCLUSIONS: Turner's syndrome patients achieve acceptable pregnancy rates after oocyte donation. A high rate of pregnancy-associated hypertensive disorders was observed which have led to a high rate of prematurity and intrauterine growth restriction. Although the number of cases in this study is limited, these results call for the need for intensive surveillance of such pregnancies. In order to reduce the risk of hypertensive disorders induced by multiple pregnancies, single embryo transfer should be proposed.     

Intratubular germ cell neoplasia in a man with ambiguous genitalia, 45,X/46,XY mosaic karyotype,and Y chromosome microdeletions

We report the case of a 17-yr-old male with ambiguous genitalia, 45,X/46,XY mosaic karyotype, and Y chromosome microdeletions. The patient underwent a testicular biopsy at the age of 6 with normal findings. A second biopsy at the age of 17 established the diagnosis of intratubular germ cell neoplasia (ITGCN), which was treated with bilateral orchidectomy. This case report deals with three important issues regarding ITGCN: First, although a prepubertal biopsy can be performed in order to provide evidence for future fertility, it is very unreliable for making a diagnosis of ITGCN. Second, because ITGCN tends to be a generalized procedure that affects both testes in a uniform pattern, a small number of biopsies, even a single one, could be adequate for diagnostic purposes in the majority of cases. Third, although the population that requires screening for ITGCN remains controversial, the early postpubertal period could be the optimum time for a testicular biopsy.     

Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen

The difficulty of identifying immature germ cells in unstained, fresh semen has led most laboratories to use the broad definition 'round cells' to indicate cells other than spermatozoa, thus grouping together both leukocytes and immature germ cells. This is also the case in research andrology, where very little attention has been given to immature germ cells in the semen apart from some rare exceptions, such as the attempts to study meiosis. Here we report on the use of a discontinuous Percoll gradient method modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa. Our technique (intra-assay variation in duplicates < 10%) demonstrated a high immature germ cell concentration in gradient fractions with 30% to 45% Percoll with a small contamination (1.5-6%) of leukocytes, confirmed by May-Grünwald-Giemsa staining, immunofluorescence and cytofluorimetry. The concentrations of immature germ cells ranged from zero in obstructive azoospermia to 2.0 x 10(6)/ml in oligozoospermia and genital tract infection. The purified immature germ cell suspensions obtained can be useful for diagnostic and research purposes.     

Endometrioma of uterine serosa in a woman with mosaic Turner's syndrome receiving hormone replacement therapy: case report

Endometriosis in Turner's syndrome patients has only been reported in five isolated cases. We present here an endometrioma on the uterine serosa and pelvic endometriosis arising in a mosaic Turner's patient receiving hormone replacement therapy (HRT). The 24 year old patient with mosaic Turner's syndrome [45,X; 46,X pseudo dicentric Y (q11.23)], on cyclic HRT after laparoscopic gonadectomy 5 years previously, was found to have an adnexal mass on routine examination. Given her history, due to the fear of a malignant process arising from a potential gonadal remnant, she underwent a laparoscopy and was found to have a 5 cm serosal endometrioma arising on a stalk from the uterine fundal surface as well as pelvic endometriosis. De-novo endometrioma and endometriosis occurred in a mosaic Turner's patient after gonadectomy on cyclic HRT. The presentation was also unusual with a pedunculated endometrioma arising from the uterine serosa. Due to the fact that the patient did have cyclic menstrual flow, her endometriosis may have arisen from retrograde menstruation or coelomic metaplasia induced by exogenous hormones.     

Involvement of E-cadherin and beta-catenin in germ cell tumours and in normal male fetal germ cell development

Intercellular contacts, mediated by E-cadherin, are essential for germ cell migration and maturation. Furthermore, it has been suggested that decrease or loss of E-cadherin correlates with tumour progression and invasive behaviour. beta-catenin is involved in a number of different processes, including cell--cell interaction when bound to cadherins, and determination of cell fate in pluripotent cells when activated via the Wnt signal-transduction pathway. To shed more light on the role of these factors in normal fetal germ cell development and the pathogenesis of germ cell tumours (GCTs), the present study investigated the presence and localization of E-cadherin and beta-catenin by immunohistochemistry. E-cadherin was only weakly expressed in or absent from fetal germ cells of the second and third trimesters, and was not expressed in carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU) and gonadoblastoma, the precursor of an invasive GCT in dysgenetic gonads. In GCTs, it was generally not expressed in seminoma and dysgerminoma, but was found in the vast majority of non-seminoma cells. beta-catenin was found in the cytoplasm of fetal germ cells at all gestational ages and in spermatogenesis in post-pubertal testes. It was also present in CIS/ITGCNU and gonadoblastoma. Whereas seminomas and dysgerminoma were negative, non-seminoma cells were frequently found to express beta-catenin. Expression of both factors therefore reflects the degree of differentiation of these tumours. No differences for either E-cadherin or beta-catenin were observed between samples of tumours resistant or sensitive to chemotherapy, and E-cadherin expression did not correlate with vascular invasion. E-cadherin and beta-catenin therefore play a role in both normal and malignant germ cell development and differentiation that warrants further investigation, but they seem to be of limited value as predictive or prognostic factors in GCTs.     

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours

AIMS: NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens. METHODS AND RESULTS: We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis. CONCLUSIONS: NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.     

High-throughput microRNAome analysis in human germ cell tumours

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow inter-sample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs.     

The role of nitric oxide in the apoptosis of neurons in the retina of the human fetal eye

The locations of NADPH-diaphorase (NADPH-d), inducible NO synthase (iNOS), and TUNEL-immunoreactive neurons in the retina of human fetuses collected during the first to third trimesters of pregnancy were studied. High levels of NADPH-d activity were seen in the inner segments of light-sensitive cells, amacrine cells, and ganglion cells. The population of NADPH-d-positive amacrine cells included three types of neuron. Type 1 neurons were large and had sparse dendritic fields occupying the inner nuclear and outer retinal layers. Small type 2 neurons were located in the inner retinal layer. Ectopic amacrine cells, type 3, were located in the outer part of the ganglion layer. A high density of NADPH-d-positive neurons was seen in the central part of the retina, surrounding the central fovea and optic disk area. NADPH-d activity increased progressively during ontogenesis and correlated with the appearance of immunoreactive iNOS in neurons. iNOS labeled a subpopulation of amacrine and ganglion cells, which appeared at 20–21 weeks of development and reached a peak of immunoreactivity by the end of the third trimester. TUNEL-immunopositive neuron nuclei with signs of apoptotic destruction were seen at 30–31 weeks of pregnancy. The greatest apoptotic index was seen in the ganglion and amacrine cell populations. These data identify NO as a factor mediating apoptosis of neurons during the critical period of differentiation of interneuronal connections in the human retina. Director: Doctor of Biological Sciences M. A. Vashchenko Director: Doctor of Biological Sciences S. L. Kondrashov __________ Translated from Morfologiya, Vol. 129, No. 1, pp. 42–49, January–February, 2006.     

Second-trimester maternal urine human chorionic gonadotrophin beta-core fragment concentrations in Asian pregnancies with fetal chromosomal abnormalities.

The aim of this study was to investigate the second trimester concentrations of maternal urine human chorionic gonadotrophin beta-core fragment (HCGbetacf) in Asian pregnanci2es with fetal chromosomal abnormalities. HCGbetacf concentrations were analysed from 34 urine samples in chromosomally abnormal pregnancies, including 28 cases of Down's syndrome, one case of trisomy 18, and five cases of other chromosomal abnormalities (one mosaic deletion and four translocations), and in a cohort of 268 normal pregnancies receiving second trimester amniocentesis. Results were normalized to urine creatinine (Cr) concentration and converted to the multiple of the median (MOM) concentration for the appropriate gestation. The median HCGbetacf MOM concentrations of Down's syndrome pregnancies (12.89) was significantly higher than that of normal pregnancies (1. 06) (P < 0.00001). Wide variations of HCGbetacf concentrations were observed in other chromosomally abnormal pregnancies. There were 18 of 28 (64%) Down's syndrome cases but one of five (20%) other chromosomally abnormal cases with HCGbetacf concentrations above the 95th centile of the control values (8.22 MOM cut-off). These findings suggest that HCGbetacf could be a potential marker in urine screening for fetal Down's syndrome in Asians.     

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis,inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer''s disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.     

Signals and molecular pathways involved in apoptosis, with special emphasis on human endometrium

Apoptosis is a selective process for deletion of cells in variousbiological systems. This event, similar to proliferation, istightly regulated, with both processes playing essential rolesin the homeostasis of renewable tissues. In human endometrium,proliferation and apoptosis occur at opposing poles of the menstrualcycle. The proliferative phase is marked by rapid growth ofthe endometrial epithelial lining, whereas progressive increasein apoptosis in this tissue is the hallmark of the secretoryand menstrual phases. The purpose of this review is to highlightsome of the signals and molecular events which are associatedwith and that may participate in apoptosis. This is followedby a review of the current literature regarding apoptosis inhuman endometrium.     

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome‐related antigen A (SSA) and La/SSB in salivary gland epithelial cells

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren’s syndrome (SS) autoantigens Ro/Sjögren’s syndrome-related antigen A (SSA) and La/Sjögren’s syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.