Establishment of human endometrial cell cultures

Epithelial and stromal endometrial cells from 19 patients atdifferent phases of the menstrual cycle were enzymatically separated,isolated by successive centrifugation and primary cultures establishedfor in-vitro studies on implantation. The behaviour of cellsin vitro was evaluated using Nomarski's inverted optics, May-Grunwald-Giemsastained coverslips and scanning electron microscopy. Epithelialand stromal cells from all patients grew successfully in Chang'smedium and formed a mixed confluent monolayer of epithelloidand fibroblastic cells in 3–7 days and such monolayerscould be maintained alive up to 3–4 weeks. Epithelioidcells were polyhedral and grew as islands in a whorl-like wavypattern around glandular fragments. Fibroblasts were spindleshaped,more long-lived and grew rapidly to form parallel bundles ofcells. Significant differences were observed in the number ofmultinucleated cells and cells with intracytoplasmic vacuolesbetween endometrium from proliferative, postovulatory and secretoryphases (P < 0.01). Scanning electron mlcrographs showed cellswith cilia with varying densities of microvilli and apical protrusions.Endometrial cells in culture showed structural features remarkablysimilar to those described for cells in situ. The method describedallows the propagation in vitro of separate endometrium celltypes which can be used to study implantation mechanisms inunstiniu lated and stimulated cycles.     

Establishment of human ampullary cell cultures

Epithelial cells from the ampulla of healthy ovlducts from 16women aged 33–41 years and at different phases of theirmenstrual cycles were used to establish primary cultures andthe continuation of a cell line. The morphology and behaviourof these cells in vitro were evaluated using Nomarski's invertedoptics, scanning and transmission electron microscopy. Cellsfrom all patients produced confluent monolayers in 6–7days with no significant relationship of cell growth with stageof cycle. Fourteen primary cultures were of the epithelioidtype while two showed mixed eplthelioid and fibroblast-likegrowth. Two distinct cell types (ciliated and secretory) wereobserved in primary culture. Secretory cells showed severalmicrovilli of different lengths and distribution. Secretorycells predominated over ciliated cells In all patients, butmaximum ciliation occurred around the time of ovulation. Structuralfeatures of the cells in vitro were remarkably similar to thosedescribed in vivo. Ampullary cells could be maintained in vitrothrough four to six passages with 3–4 days of growth betweenpassages. Sub-cultured cells were all secretory and were oftwo types (I and II) based on ultrastructure. Secretory vesiclescontaining electron-dense material and lipids were observedin these cells. The method described allows for the use of ampullarycells as feeder layers for IVF and support of cleaving humanembryos and the evaluation of the biochemical events surroundingfertilization and ectopic pregnancies.     

Testing of nine different xeno-free culture media for human embryonic stem cell cultures

BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC. METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder cells. Cultured hESC were characterized immunocytochemically and by fluorescence-activated cell sorter analysis. The differentiation potential of hESC cultured with xeno-free media was determined with the RT-PCR analysis. RESULTS: The hESC cultured in xeno-free media differentiated or the proliferation decreased substantially. Under some test conditions, the morphology of the feeder cells was altered considerably. The hESC cultured with human serum underwent excessive differentiation in the beginning of culture, but a fraction of hESC was able to adapt to culture conditions containing 20% of human serum. CONCLUSIONS: None of the studied xeno-free media was able to maintain the undifferentiated growth of hESC. The medium containing 20% human serum was found to sustain undifferentiated hESC proliferation to some extent, yet was inferior to the conventional ko-SR-containing medium.     

The status and the state of the human epididymis

As far as can be determined now, the general organization ofthe human epididymis, the regional character of its epitheliumand luminal environment, and the maturation changes that spermatozoaundergo there are quite similar in many respects in both animalsand man. None the less, the outcomes of some clinical proceduresaimed at overcoming duct obstruction (high epididymovasostomyor micro-aspiration of some fertile spermatozoa from the caputor vasa efferentia) suggest a flexibility in the sperm/epididymalrelationship that may cast an element of doubt as to the essentialplace of the epididymis in the chain of human conception. Whilesuch clinical results make it difficult yet to be quite surethat the epididymis has the same essential sperm maturationrole in man that it does in the few animals tested, they raiseintriguing questions as to the potential ability of the humanvas deferens, and even the vasa efferentia, to contribute inthat respect. The human epididymis possesses other traits whichcollectively seem to resemble the suppressive effects of subjectinganimal epididymides to body temperature. In that regard, itis a real possibility that the elevation of scrotal temperatureimposed by clothing may be affecting in an adverse manner severalaspects of epididymal function, especially those concerned withthe storage and viability of spermatozoa, and thereby the qualityof the ejaculate.     

A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate

BACKGROUND: A novel organotypic culture system was established for modelling the hormonal responses of the normal human endometrium in vitro. METHODS: Endometrial epithelial cells were cultured as glandular organoids within reconstituted extracellular matrix (Matrigel) in tissue culture inserts and stromal cells on plastic below the epithelial compartment. The effects of estradiol (E2) and E2 together with medroxyprogesterone acetate (MPA) on cell proliferation and the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) were studied in 10 epithelial-stromal co-cultures and in three parallel monocultures of epithelial organoids. RESULTS: In co-cultures, E2 was shown to increase the percentage of Ki67-positive cells by approximately 2-fold relative to untreated controls. In the presence of MPA, a significant decrease in cell proliferation was detected. Similar results were obtained when the corresponding percentages of Ki67-positive organoids were calculated instead of individual cells. In the absence of stromal fibroblasts, Ki67 epithelial labelling remained below the control value after both hormonal treatments. Epithelial organoids retained their capacity to express estrogen and progesterone receptors in culture. E2 was shown to markedly increase and MPA to down-regulate the expression of PR. The expression of ERalpha was only slightly affected by either hormonal treatment. CONCLUSIONS: The present organotypic model provides a novel in vitro system in which to study the effects of steroids in the normal human endometrium both in terms of cell proliferation and gene expression. The culture system holds promise as a useful method to screen novel steroid compounds and may help to circumvent problems related to the use of animal models.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Monolayer culture of normal human esophageal epithelial cells

Summary A method is described for the isolation of epithelial cells from normal human esophagus and the establishment of the epithelial cells in monolayer cell culture using irradiated 3T3 fibroblast feeder layers as a substrate. The monolayer cultures were subsequently used to study the effects of a tumor promoter and carcinogen on human esophageal epithelial cells in vitro.     

Establishment of a ciliated epithelial cell line from human Fallopian tube

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had >/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (16/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/16), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/growth factors such as leukaemia inhibitory factor (LIF), interleukin (IL)-6, IL-8 and basic fibroblast growth factor were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.     

正常人乳腺上皮细胞的原代培养

目的研究正常人乳腺上皮细胞原代培养的可行性,以及激素对其增殖的影响。方法从乳房区段切除术切除组织中获取正常人乳腺组织,用胶原酶溶液消化乳腺组织分离得到较纯净的上皮细胞,用烧灼法结合酶消化法解决细胞纯化问题。细胞形态观察和电镜检测鉴定细胞。结果正常人乳腺上皮细胞的原代培养是可行的。1×10-5的雌二醇和孕酮混合液可以促进细胞增殖。     

Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization-embryo transfer success

We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle. There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied. As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point. It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro. All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies. A limited number of cycles from donor/recipient cycles did not show similar results. Steroid concentrations were not predictive of conception. These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles. This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.     

Gland and epithelium formation in vitro from epithelial cells of the human endometrium

As is often the case with cell culture, normal human endometrial epithelial cells show a low, squamous shape with flattened nuclei and lack three-dimensional morphology in in-vitro culture systems. Here we report the first well-differentiated epithelial culture system using basement membrane extracts (BME) from an Engelbreth-Holm-Swarm tumour (EHS tumour). In this model, BME regulated the reconstruction of glandular formation and subsequent reformation of the epithelium by epithelial cells. An electron microscopic study clearly indicated that there were two distinct types of cells grown on the substrate. The first, having a high columnar shape, formed the glandular epithelium, and the second, having a cuboidal shape, covered the surface of the BME. Study of epithelial reformation indicated that the regeneration of superficial epithelium could occur, following the development of the glandular formation, in a helix-like pattern. Total protein secretion was greater when the cells were grown on the BME than on plastic. Thus, normal human endometrial epithelial cells cultured on the BME assumed a phenotype and morphology characteristic of those in vivo.     

Expression of epidermal proteins resembling cytokeratin No. 2 in human prostatic epithelial cell cultures

Laboratory of Neurohormonal Regulation of Reproduction, Research Institute of Endocrinology and Metabolism, Ministry of Health of the Ukrainian SSR, Kiev. (Presented by Academician of the Academy of Medical Sciences of the USSR, I. B. Zvarskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 7, pp. 88–90, July, 1989.     

Morpho-functional study of human luteal cell subpopulations

It has been reported that the mammalian corpus luteum is composedmainly of two subpopulations of luteal cells (large and small)of different morphology and function. The aims of this studywere first to characterize cytologically the human corpus luteumthroughout the luteal phase, and second to establish the in-vitrosteroidogenic capacity of a well-defined human mid-luteal cellsystem. The results show that the most predominant (>70%)cell shape, is polyhedric, and the number of cells per unitarea is significantly different in the early, mid- and latecorpus luteum (P< 0.005). Moreover, small cells (<22 µm)were most common (56.8%) in all tissues analysed. On the otherhand, both subpopulations synthesized progesterone, oestradioland testosterone, although a significantly greater productionof basal steroids was observed in large luteal cells (P<0.05). Nevertheless, the response of small cells to human chorionicgonadotrophin (HCG) was significantly greater (P< 0.05) thanthat of large cells, in agreement with the preferential specificbinding obtained for (125I)HCG to the small cell subpopulation.In summary, these results indicate that the human corpus luteumpossesses distinct cell types, which may be related to endocrinefunction and its control.     

Exogenous steroids and the control of oestradiol secretion by human granulosa-lutein cells by follicle stimulating hormone and insulin-like growth factor-I

This study first examined the relative activities of 17-hydroxylase,17, 20-lyase and aromatase in human granulosa–lutein cellsby challenging the cells with steroid precursors in the oestradiolbiosynthetic pathway. When cells from four patients were challengedwith precursor steroids on the pathway to oestrogen synthesis(pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesteroneand androstenedione at 5 x 10^–6 M), oestradiol (nmol/l)outputs after 1 day of culture were (median, interquartile range)as follows: 4.1 (2.1– 8.8; pregnenolone), 3.1 (1.7–6.0;progesterone), 12.5 (6.9–18.1; 17hydroxypregnenolone),8.2 (4.1–16.7; 17hydroxyprogesterone) and 251 (140–819;androstenedione). No further increases were seen when the steroidconcentration was increased to 1 x 10^–5 M. Basal oestradiolsecretion was 3.5 (1.6–8.2) nmol/l. We conclude that theconversion of pregnenolone/progesterone to oestradiol by granulosa–luteincells is rate limited by 17-hydroxylase activity but that thesecells are capable of oestradiol secretion (in the nmol/l range)in the absence of androstenedione. In the second part of thisstudy we examined the control of granulosa–lutein oestradiolsecretion by follicle stimulating hormone (FSH) and insulinlikegrowth factor-I (IGF-I) in the presence and absence of exogenousandrostenedione (10^–6 M). Cells were cultured for up to6 days and basal oestradiol (nmol/l) fell dramatically overthis period both in the presence and absence of androstenedione,e.g. from 339 (223–419) (median and interquartile range,cells from five patients cultured in the presence of androstenedione)after 2 days to 14 (7–59) after 6 days. There was no effectof FSH (83/575, 0–160 IU/l) in the absence of androstenedionebut in its presence FSH (96 IU/l) increased oestradiol secretionslightly by 153 ±45 (day 4) and 151 ± 21 (day6; results mean ± SEM, percentage increase over time-matchedcontrols; cells from five patients). In contrast, the effectof IGF-I (30 ng/ml) was to markedly enhance oestradiol secretionto 1099±320% (n = 7) of the control (day 4) in the absenceof exogenous androgen and to 551 ± 184% (n = 5) in itspresence. There was no evidence of any synergistic interactionbetween IGF-I and FSH during the culture period in the absenceof androstenedione. However, there was a synergistic effectfor IGF-I (30 ng/ml)/FSH (96 IU/l) after 6 days in culture inits presence in that oestradiol secretion increased by 1748± 294% (n = 5) compared to 157 ± 21% (FSH) and1211 ± 233% (IGF-I) for the stimulators on their own.However, this effect may be explained, in part, by the increasein cell number provoked by IGF-I over this culture period. Weconclude that (i) under these conditions granulosa cells showedlow 17-hydroxylase activities, (ii) granulosa cells are capableof synthesizing oestradiol in the absence of exogenous androgens,the substrates for the aromatase complex, and (iii) FSH is oflittle importance in stimulating oestradiol secretion from granulosa-luteincells, and the evidence for it positively modulating IGF-I activityis poor.     

Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Papillary cystadenoma of the epididymis is an uncommon benign lesion that may occur sporadically or as a manifestation of von Hippel—Lindau (VHL) disease. Neither immunohistochemical studies nor molecular genetic analyses of the VHL gene have been reported previously for this lesion. The authors describe two cases of clear cell papillary cystadenoma of the epididymis, both of which were initially confused with metastatic renal cell carcinoma. Both lesions showed positive immunohistochemical staining for low and intermediate molecular weight keratins (Cam 5.2 and AE1/AE3), EMA, vimentin, α₁-antitrypsin, and α₁-antichymotrypsin. Each was negative for CEA. Because clear cell papillary cystadenoma is similar to renal cell carcinoma histologically, and because both occur as components of the von Hippel—Lindau disease complex, the authors analyzed both cases for the presence of mutations in the VHL gene. A somatic VHL gene mutation was detected in one of the two tumors by polymerase chain reaction followed by single-strand conformation polymorphism analysis. Direct sequencing revealed a cytosine to thymine transition at nucleotide 694, resulting in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. As the VHL gene is believed to function as a tumor suppressor gene, VHL gene mutations may play a role in the initiation of tumorigenesis in sporadic cystadenomas of the epididymis.     

The effect of progesterone upon first trimester trophoblastic cell differentiation and human chorionic gonadotrophin secretion.

The effect of progesterone (P) upon first trimester placental secretion of human chorionic gonadotrophin (HCG) and cellular differentiation was studied using both static and kinetic methods. At 1 microM, P inhibited spontaneous episodic secretion of HCG when given in short pulses (1-4 min) to placental explants in superfusion. Both HCG pulse frequency and amplitude were reduced. At 0.1-0.01 microM P concentrations, the effect of HCG secretion was milder. P also blocked the maximally effective concentration 100 pM of gonadotrophin releasing hormone (GnRH) analogue, a known HCG stimulant, when given together with it for 1 min. This inhibitory effect lasted for 1 h after P administration. Progesterone at 1 microM, added daily for 1 week blocked HCG secretion by isolated trophoblastic cells in static culture. This inhibitory effect lasted until the fifth day. No effect on differentiation and long-term viability was noticed in P-treated cells. Incubation with 0.1-1.0 microM P did not affect HCG secretion by explants after 24 h. In contrast, the effect of 1 microM cortisol or 1 nM oestradiol was stimulatory. In conclusion, P exerts both a rapid and delayed inhibitory effect upon HCG secretion and production. It may do so by counteracting the stimulatory effect of endogenous GnRH on gonadotrophin secretion by the placenta.     

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro.

Micromanipulation of human oocytes and embryos has provided new opportunities for both the treatment of infertility and the preimplantation diagnosis of genetic disease. It is important to determine whether manipulated embryos develop normally in vitro, as an indication of their suitability for transfer. However, at present there is little information on the development of non-manipulated embryos in vitro for comparison. We have therefore monitored morphological changes and human chorionic gonadotrophin (HCG) secretion in 36 non-manipulated human embryos, including 26 blastocysts and 10 cavitating morulae, daily from day 3 to day 14 of culture. Hatching was observed in 10 (38.5%) blastocysts and five of these adhered to the culture dish and appeared viable until day 14. The secretion of HCG was first detected on day 8, peaked at day 10 (51.11 +/- 8.7 mIU/ml) and then declined but was still detectable in four blastocysts on day 14. There was no overall difference in HCG secretion by hatched blastocysts and those which remained within the zona. However, those hatched blastocysts which showed adherence had significantly increased (P less than 0.05) HCG secretion. For individual blastocysts, the pattern of HCG secretion correlated well with the assessment of morphology. These data provide the basis for comparative studies of morphological changes and HCG secretion in manipulated embryos.     

Expression of melatoninergic receptors in human placental choriocarcinoma cell lines

BACKGROUND: Melatonin crosses the placenta and enters the fetalcirculation. Moreover, experimental data suggest a possibleinfluence of melatonin on placental function and fetal developmentin humans. To date, the expression and role of melatonin receptorsin human placenta choriocarcinoma cell lines and in human termplacental tissues remain to be elucidated. METHODS AND RESULTS:Results from RT–PCR, western blotting and confocal microscopydemonstrated that the MT1, MT2 and ROR1 melatonin receptorsare expressed in the human term placental tissues and in choriocarcinomacell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbentassay showed that 6-chloromelatonin (a melatonin agonist) inhibits,in a dose-dependent manner, forskolin-stimulated hCG- secretionin JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but hadno effect on basal human chorionic gonadotrophin (hCG-) levels.This effect of 6-chloromelatonin on forskolin-stimulated HCG-secretion was abolished by pertussis toxin (PTX), suggestingthat melatonin regulates hCG- production by an action involvingan inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatoninstimulated basal hCG- secretion (P < 0.001). CONCLUSION:These results demonstrate, for the first time, the expressionof melatonin receptors in human term placental tissues and inchoriocarcinoma cells and suggest a possible paracrine/autocrinefunction for melatonin in human placenta.