Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Changes in calcium concentrations in subcellular compartments of rat submandibular gland acinar cells induced by cholinergic stimulation

 The distribution of anti-Müllerian hormone (AMH) and laminin (Ln) α5 chain in differentiating rat testis and ovary were studied by immunohistochemistry. In the incipient embryonic male gonad a weak reaction for Ln α5 chain, but not for AMH, was detected. With further prenatal development, Ln α5 chain rapidly disappeared from the basement membrane (BM) of the incipient testicular cords in parallel with the appearance of AMH in the Sertoli cells. After birth, Ln α5 chain reappeared in the BMs of the cords with the decline and disappearance of AMH from the respective Sertoli cells. In the corresponding stages of the ovary, Ln α5 chain was present in the BM of the prenatal gonadal cords and in postnatal primordial follicles. The cells of those epithelia were negative for AMH. With the growth of the follicles, Ln α5 chain disappeared from the BM when AMH appeared in the epithelial follicular cells. The present results show that male and female gonadal epithelia negative for Ln α5 chain were positive for AMH, and that epithelia positive for Ln α5 chain were negative for AMH. Thus, epithelial Ln α5 chain and AMH as a product of the same cell seemed to exclude each other. The results require an explanation why Ln α5 chain has to be excluded from the BM of the epithelia during the secretion of AMH chain through the basal cell membrane to the surrounding tissues where it executes its important biological functions. These observations suggest a hypothesis that the production of both components is regulated by the same gene and factor system. Accepted: 14 January 1999     

Influence of age and medium on formation of epithelial cords in the rat fetal ovary in vitro

Fetal ovaries of 14.5-day-old rats were cultured for periods of up to 19 days in control medium or in medium conditioned by the preliminary culture of testes from fetal or young rats. In all ovaries, after 12 days of culture in either medium, epithelial cords were noted having an aspect identical to that of seminiferous cords present in fetal testes explanted at 14.5 days and also cultured for 12 days, i.e. the epithelial cords appeared in ovaries when there was no 'male' or testicular influence. The appearance of histological preparations suggested that the disappearance of the germ cells might bring about a reorganization of the follicular cells in epithelial cords during the differentiation period of the first follicles. With ovaries cultured in conditioned medium, degeneration of the germ cells was more marked, follicles were rare and intra-ovarian cords were greater in number than in ovaries cultured in control medium. The ovaries thus transformed produced the anti-Müllerian hormone (AMH) although they lacked the "germinostatic activity" normally developed by testes of fetal or young rats. This germinostatic activity prevents the multiplication of oogonia when the testes and ovaries are co-cultured in vitro. The transformed ovaries therefore do not have all the functional capacities of fetal testes.     

Reaggregates of cells from rat testis resemble developing gonads

Reaggregates prepared from newborn rat testis cells in Moscona-type rotation cultures were analyzed and compared with normal fetal (12-21 days) and newborn testes at the light and electron microscope level. After 25 h of culture, the aggregates resembled normal testicular tissue. The cells of the surface layer were spindle-shaped and connected by adherent junctions. The epithelial cords were composed exclusively of Sertoli cells and were surrounded by elongated cells resembling the developing myoid cells in newborn testes. The basal aspect of the cords was covered by a layer of flocculent material which, in places, was organized like an ordinary basement membrane. Individual spermatogonia with pseudopodes were observed in the interstitial tissue. Some Leydig cells were organized into small clusters like those typical in newborn testes. The present observations indicate that, histologically, the reaggregation of separated testicular cells resembles the differentiation of embryonic male gonads.     

Requirement of serum components for the preservation of primordial germ cells in testis cords during early stages of testicular differentiation in vitro in the mouse

Mouse gonadal primordia were isolated from embryos on the 11th day of gestation and cultured in vitro. They developed into either testes or ovaries after 7 days of culture in Eagle's minimum essential medium (MEM) supplemented with horse serum, whereas they did not differentiate in MEM alone. We studied how serum components are required for testicular development in vitro. When gonadal primordia were cultured in MEM alone for the first 1-3 days and subsequently in MEM supplemented with serum, testis cords developed while germ cells disappeared or only a few remained in the testis cords. In contrast, when serum was present in the medium during the first day of culture and omitted thereafter, germ cells were retained within testis cords. These results suggested that some serum component(s) is specifically required by germ cells independent of testis cord organization. Of more than 10 serum components tested, low and very low density lipoprotein fractions increased the number of germ cells in testicular explants.     

Ultrastructure of the early ovary and testis in pig embryos.

Pig embryos aged 26-27 days were used for an ultrastructural study of the early ovary and testis. Sex was identified by both chromosomal analysis and gonadal histology, with consistent results. The gonads occupied their original site in the medial coelomic angles in both sexes. The female gonad was composed of three tissues: the surface epithelium, the gonadal blastema and the mesenchyme. The gonadal structure was similar to that seen earlier at the age of 24 days. At 26 days the testis had distinctly differentiated into four tissues. The new components were the testicular cords and the interstitium, both derived from the gonadal blastema. The testicular cords resembled anastomosing sheets more than cords. The ultrastructure of the tissues and their cell types are described and compared to the previous indifferent stage at the age of 24 days. The cells of the surface epithelium, of the primitive cords, of the mesenchyme, and the primordial germ cells had an ultrastructure that was similar in both sexes. The sustentacular cells of the testicular cords resembled the primitive cord cells and the spermatogonia were similar to the primordial germ cells. No Leydig cells were present yet. The process of testicular differentiation is described on the basis of the present and a previous study, and a new hypothesis, based on the vascular organization, is presented.     

Intermediate filaments and epithelial differentiation of male rat embryonic gonad

The presence and distribution of desmin, vimentin, cytokeratin, and laminin in the gonads of developing male rat embryos (11-17 days) were studied by immunocytochemistry. The findings were correlated with morphological changes of the cells and with the formation of basement membranes, as determined by electron microscopy. The surface epithelial and subepithelial cells of the meesonephros in the prospective gonadal region contained desmin. At the onset of gonadal development, vimentin appeared in the somatic cells of the thickening surface epithelium, which formed the gonadal ridge. Desmin disappeared and cytokeratins appeared in the Sertoli precursor cells at the inception of their epithelial differentiation. Simultaneously, the prospective Sertoli cells became polarized during their assembly into epithelial cell aggregates; the aggregates then fused and formed elongated testicular cords. The epithelial cell differentiation was accompanied by a deposition of basement membrane material around the cords and by an increase of desmin in the cells immediately around the cords. With further differentiation of the testicular cords, some cytokeratins from the Sertoli cells, but not from the cells of the rete cords, disappeared. On the other hand, other cytokeratin polypeptides and vimentin remained in the fetal Sertoli cells. The surface cell layer slowly differentiated towards a proper epithelium after the basic formation of the testicular cords and interstitium. Desmin and vimentin persisted in the interstitial cells throughout the entire study period. The early differentiation of the gonad is apparently under a general sex-independent initiation program. The developmental changes in intermediate filaments offer an opportunity for the further analysis of their general role in early organogenesis. In light of the genetic theory of testicular differentiation, the functions of the regulatory factor(s) include specific organization of cord cells, histological organization into looping cords rather than separated follicles, and male development of the interstitium, surface epithelium and tunica albuginea.     

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.     

Establishment of propagable epithelial cell lines from normal adult rat pancreas

A method for establishing propagable epithelial cell lines from normal adult rat pancreas is described. Morphological studies showed that these cells were derived from duct epithelial cells. These cells grew equally well in media containing fetal bovine (FBS) or horse serum (HS). Preliminary studies suggested that propagable cultured pancreatic ductal cells during early passages retained some capacity to differentiate into acinar-like cells with the formation of granules resembling zymogen, especially when these cells were cultured on mixed ester cellulose membrane. This supports the concept that pancreatic ductal lining cells represent the 'stem' cells on pancreatic epithelial cells. Propagable pancreatic epithelial cells in long-term cultures will be useful in the histogenetic and mechanistic studies of pancreatic carcinogenesis.     

Anti-Müllerian hormone: Hormone or growth factor?

Anti-Müllerian hormone (AMH) is a dimeric glycoprotein member of the TGF-β family. It is synthesized by immature Sertoli cells, and, to a lesser degree, by adult Sertoli and granulosa cells. AMH is responsible for the regression of Müllerian ducts in the male fetus; it also has deleterious effects on the female fetal reproductive tract, destroying Müllerian primordia and germ cells, and masculinizing the fetal ovary on the rare occasions female fetuses become exposed to its effects. All other suggested actions for AMH—retardation of oocyte meiosis, inhibition of EGF receptor autophosphorylation, anti-cancer activity—have been reported with crude hormone preparations, and have not been confirmed using pure AMH. Its relatively limited sphere of action—the fetal genital tract—and the fact that it is secreted into the general circulation and can act at long range, imply that AMH is more like a hormone than a growth factor, but the complex interaction between hormones and growth factors make a formal distinction impossible.     

Early stages of testicular differentiation in the rat

Summary The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells show an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords.The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.     

Use of monoclonal antibody techniques to study the ontogeny of bovine anti-Müllerian hormone

Monoclonal antibodies against bovine anti-Müllerian hormone (AMH) were used to study the hormone in cattle. Anti-Müllerian activity of testicular tissue, immunoreactive testicular AMH, serum AMH concentration and AMH production by incubated testicular tissue were detectable from 42 days, i.e. at the time of seminiferous tubule differentiation, and peaked between 50 and 80 days, when the Müllerian ducts regress in the male fetus. All the values stabilized at a lower level until 30 days after birth and then slowly decreased. At 18 months, only traces of AMH immunoreactivity were detectable in testicular tissue and serum concentration and AMH production by incubated testicular tissue were negligible; the main source of AMH in the adult animal was the rete testis fluid. Study of the disappearance rate of AMH from the serum of castrated calves gave a half-life of approximately 2 days for bovine AMH.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

New insights on the mechanism of testis differentiation from the morphogenesis of experimentally induced testes in genetically female chick embryos

Embryonic testes grafted in the extraembryonic coelom of 3-day-old genetically female chick embryos may induce total and definitive reversal of gonadal sex differentiation. In this experimental condition, the left gonad becomes a testis instead of an ovary. This makes it possible to compare testicular and ovarian morphogenesis in animals having the same genetic sex and to discount what is due to differences in the genetic determination between male and female. The morphogenesis of such testes is marked by a disappearance of the cortical germinal epithelium. The medullary sex cords keep a narrow lumen instead of becoming large lacunae. The germ cells remain few in the sex cords and do not become meiotic. Furthermore, interstitial cell development is known to be very slow. As a consequence the gross size of the gonad is much smaller than that of an ovary. All these morphogenetic phenomena are unlike those observed during normal ovarian differentiation and evidence an inhibiting influence of the grafted testes. Since inhibition and masculinization are concomitant, inhibition appears to be the mechanism responsible for gonadal sex reversal. The extraembryonic situation of the grafted testes and their relation with the embryo only via the blood stream demonstrates the role of a secreted substance or substances still to be exactly identified. Previous data suggest that this could be the anti-Müllerian-hormone (AMH). Furthermore, previous and present results show that testis differentiation can be actively induced in a bird. This does not agree with the hypothesis that the gonads of the homogametic sex, i.e., the testes in birds, do not need any inducer in order to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The presence of a common embryonic blastema for ovarian and testicular parenchymal (follicular,interstitial and tubular) cells in cattle,Bos taurus

Summary The early embryonic gonadal development in the cattle is characterized by the appearance of an alkaline phosphatase positive blastema. Its derivatives in gonads of both sexes, follicular cells in the female and interstitial cells in the male, also show positive alkaline phosphatase reaction. Primordial germ cells are equally alkaline phosphatase positive, but loose this activity when they later transform to oögonia and oöcytes, or to spermatogonia respectively. Using the enzyme activity as label to trace these constituents in the developmental steps of the bovine gonads, the following results were obtained.Differentiation processes leading to the appearance of the sex cords take place in situ within the gonadal blastema which occupies the main central part of the gonadal fold. It is essentially a segregation process of the follicular cell cords or of the interstitial cells and the tubular primordia from the undifferentiated common anlage.The so-called germinal epithelium is not involved in the differentiation of sex cords. Its participation — if any — in the gonadal development is restricted to a very short and rather early period. Secondary sex cords (Pflügers cords) do not occur. In the cattle there is no reason to assume a cortico-medullary antagonism in the sex determined gonadal development.It can be assumed that the follicular cells in the ovary and the interstitial cells in the testis are homologous. This applies possibly also to the tubular cells of the testis. Homology should be admitted also for the rete structures, which remain small and undeveloped in the ovary while in the male they show considerable development.In the ovary the follicular cell cords differentiating within the central blastema match in a junctional zone with the peripheral layer of oögonia. These are taken up by the most peripheral branches of the follicular cell cords, thus transforming to ovigerous cords. During the downward movement within these cords the germ cells transform to oöcytes which for their part proceed through first meiotic prophase and reach the dictyotene stage. The maturation of the germ cells seems to be controlled by the follicular cells and may even temporarily get out of control until an adequate number of follicular cells is found in vicinity of individual oöcytes to form primordial follicles.The alkaline phosphatase reaction reveals the presence of numerous persisting remnants of follicular cell cords in the developing and even adult ovary.It is suggested that the findings in the cattle gonads can be applied also to other mammals, mainly to those with longer gestation periods like man.Contribution No 58-66, Department of Biology, City of Hope Medical Center. This work was supported in part by a grant (CA 05138) from the National Cancer Institute, U.S. Public Health Service. The project was undertaken during a five-month visit to Dr. Ohno's laboratory by the senior author whose expenses were covered by the Deutsche Forschungsgemeinschaft.     

A quantitative and interspecific test for biological activity of anti-müllerian hormone: the fetal ovary aromatase assay.

Anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor, has previously been shown to sex-reverse the steroidogenic pattern of fetal mammalian ovaries through repression of aromatase biosynthesis. Study of the ontogeny of the response of cyclic AMP-stimulated aromatase activity of rat fetal ovaries to AMH has allowed us to develop a quantitative bioassay for the hormone. Linear responses as a function of the logarithm of AMH concentration were observed over ranges of 0.2-7.5 micrograms/ml for the bovine protein and 0.15-2 micrograms/ml for the human protein, with a maximal decrease in aromatase activity of 90% for both proteins. Under the same in vitro conditions, AMH treatment did not affect cyclic AMP-stimulated fetal rat testicular aromatase activity. Partially purified chick AMH also decreased rat ovarian aromatase activity, allowing us to use this test to study AMH ontogeny in chick gonads. Analysis of the species specificity of AMH repression of ovarian aromatase activity indicated that turtle and rat fetal ovaries responded to AMH of other vertebrate classes, whereas aromatase activity of chick embryo ovaries could be repressed only by the homospecific hormone.