Wolffian duct differentiation by physiological concentrations of androgen delivered systemically

In developing mammalian males, conversion of the Wolffian ducts into the epididymides and vasa deferentia depends on androgen secretion by the testes, whereas in females these ducts remain in a vestigial form or regress. However, there is continuing uncertainty whether the androgen needs to be delivered locally, either by diffusion from the adjacent testis or, by secretion into the lumen of the duct, or whether circulating androgens maintain and virilize the Wolffian ducts. To resolve this uncertainty, we transplanted either day 0-2 or day 8-9 post-partum testes beneath the flank skin of three groups of neonatal (days 0-1) female tammar wallabies, where they developed and secreted physiological levels of hormones. The Wolffian ducts of all these females were retained and had formed extensive epididymides when examined at days 25, 34 and 87 after birth. In the two older groups of females, sampled after the time of prostatic bud formation, the urogenital sinus was virilized and there was extensive prostatic development similar to that of normal males of the same age, showing that androgen secretion had occurred. Virilization of the Wolffian ducts occurred during an early but short-lived window of sensitivity. This study provides the first clear evidence that under physiological conditions virilization can be mediated by circulating androgen.     

Absence of sex differences in size of the genital ducts of the rat prior to embryonic day 15.5-16.0.

Sexual dimorphisms of the rat brain are generally believed to be brought about by the presence of testosterone during a critical period starting at embryonic day (ED) 17/18. In contrast, sex differences of diencephalic and mesencephalic dopaminergic neurons were observed to develop in cell cultures raised from ED 14 rat brains. This was interpreted as evidence indicating that sexual differentiation of certain neural systems may occur independently of gonadal hormones. To substantiate this claim, it was felt necessary to examine the rat embryo for clues to a possible existence of sex differences in hormonal environment prior to ED 17. Morphometry was applied to compare the development of male and female Wolffian and Müllerian ducts, both primary targets of hormones secreted from the male gonad. Diameters of serially cross-sectioned Wolffian and Müllerian ducts were measured in rats of ED 15.0 to ED 16.5. Females had thicker Müllerian ducts from ED 15.5 on. The first step of differentiation in males was the widening of the lumen and a slight increase of the outer diameter of the Wolffian duct at ED 16.0. The size differences of both ducts were most obvious in the vicinity of the lower half of the gonad. Except in Wolffian ducts of ED 16.5, sex differences were absent in the caudal parts of the ducts. It appears that gonadal androgen and Müllerian inhibiting substance do not affect the development of their classical target organs prior to ED 16.0 and ED 15.5, respectively. Furthermore, the first effects are paracrine in nature. There is no evidence for sex differences in systemic androgen environment until ED 16.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens and the development of the vagina

Today it is generally held that the vagina develops from sinovaginal bulbs and that the lower third of the definitive vagina is derived from the urogenital sinus. Here we show that the entire vagina arises by downward growth of Wolffian and Müllerian ducts, that the sinovaginal bulbs are in fact the caudal ends of the Wolffian ducts, and that vaginal development is under negative control of androgens. We designed a genetic experiment in which the androgen receptor defect in the Tfm mouse was used to examine the effects of androgens. Vaginal development was studied by 3D reconstruction in androgen-treated female embryos and in complete androgen-insensitive littermates. In androgen-treated females, descent of the genital ducts was inhibited, and a vagina formed in androgen-insensitive Tfm embryos as it does in normal females. By immmunohistochemical localization of the androgen receptor in normal mouse embryos, we demonstrated that the androgen receptor was expressed in Wolffian duct and urogenital sinus-derived structures, and was entirely absent in the Müllerian duct derivatives. We conclude that the Wolffian ducts are instrumental in conveying the negative control by androgens on vaginal development. The results are discussed under evolutionary aspects at the transition from marsupial to eutherian mammals.     

Müllerian inhibiting substance in reproduction and cancer.

During embryogenesis normal male phenotypic development requires the action of Müllerian Inhibiting Substance (MIS) which is secreted by Sertoli cells of the fetal testis. As testes differentiate in genetic (XY) males, they produce MIS which causes regression of the Müllerian ducts, the anlagen of the female reproductive tract. Soon thereafter, testicular androgens stimulate the Wolffian ducts. In females, on the other hand, MIS is not produced by grandulosa cells until after birth, before which, estrogens induce Müllerian duct development, while the Wolffian ducts passively atrophy in the absence of androgenic stimulation. High serum MIS levels in males are maintained until puberty, whereupon they fall to baseline levels. In females MIS is undetectable in serum until the peripubertal period when values approach the baseline levels of males. This distinct pattern of sexual and ontogenic expression presupposes and requires tight regulation. MIS may play a role in gonadal function and development. Our laboratory has shown that an important role for ovarian MIS is to inhibit oocyte meiosis, perhaps providing maximal oocyte maturation prior to selection for ovulation and subsequent fertilization. Furthermore, Vigier et al. (Development 100:43-55) have recently obtained evidence that MIS may influence testicular differentiation, coincident with inhibition of aromatase activity. Current structure-function studies demonstrate that MIS, like other growth regulators in its protein family, requires proteolytic cleavage to exhibit full biological activity. MIS can be inhibited by epidermal growth factor. This antagonism, which is common to all MIS functions so far investigated, is associated with inhibition of EGF receptor autophosphorylation. We have provided evidence that bovine MIS can inhibit female reproductive tract tumors arising in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice

The distribution of nuclear oestrogen receptor alpha (ER-alpha) in the sex organs of fetal and newborn mice has been investigated immunohistochemically. There was no visible ER-alpha immunoreaction in the sexually undifferentiated gonads, whereas a faint immunoreaction was detected in a few cells surrounding the sex ducts, the Müllerian and Wolffian ducts. After sex differentiation, the immunoreaction of ER-alpha was observed in various cell types, with the exception of both the male and female germ cells. In the fetal ovary, immunoreaction was restricted to the surface epithelium and a few stroma cells without any preferential localization. In the testis, the number of ER-alpha-immunopositive cells, identified as Leydig and peritubular cells, increased with age. Immediately after sex differentiation, cells surrounding the sex ducts were ER-alpha-immunopositive, but no immunoreaction was detected in the epithelium in either sex. During development, the epithelium of the sex ducts attained a topographic difference in ER-alpha immunoreaction. In females, immunoreaction was detected in the epithelium of the oviduct, but not in the uterus. In males, the immunoreaction of ER-alpha was intense in the epithelium of the efferent ducts, weak in the epididymis and absent in the vas deferens. ER-alpha immunoreaction in the cells surrounding the sex duct differed between the sexes, being high in all these cells in females, but of varying intensity in males. ER-alpha may not play an important role in the development and function of ovarian cells, but may be involved in the development of Leydig and peritubular cells. Furthermore, substances that react with ER-alpha may influence the male germ cells indirectly through the ER-alpha-immunopositive peritubular cells. In addition, in both sexes, ER-alpha-immunopositive cells surrounding the sex ducts may be involved in the mediation of growth and functional differentiation of the ductal epithelium.     

Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females

The whole-body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was significantly higher on Fetal Days 18 and 19 in male than in female rats; androgen content was equivalent in the two sexes at other fetal ages, including Days 16, 17, 20, and 21, and prior to parturition on Fetal Day 22. These results partially corroborate previous data of Weisz and Ward (Endocrinology 1980; 106:306-316), who measured testosterone in pooled plasma from rat fetuses and suggest that androgens contribute to masculine brain sexual differentiation only briefly during fetal life. No significant differences in whole-body androgen content were observed among groups of females situated in utero between 0, 1, or 2 males on each side (contiguous male model) or among groups of females with 0, 1, or 2 or more males located caudally (on the cervical side) in the same uterine horn, regardless of whether combined data from Fetal Days 17-22 or only Fetal Days 18 and 19 were considered. These results provide no evidence that androgens from males reach female fetuses in the same uterine horn.     

Male disadvantage? Fetal sex and cardiovascular responses to asphyxia in preterm fetal sheep

Clinically and experimentally male fetuses are at significantly greater risk of dying or suffering injury at birth, particularly after premature delivery. We undertook a retrospective cohort analysis of 60 female and 65 male singleton preterm fetal sheep (103-104 days, 0.7 gestation) with mean arterial blood pressure (MAP), heart rate, and carotid and femoral blood flow recordings during 25 min of umbilical cord occlusion in utero. Occlusions were stopped early if fetal MAP fell below 8 mmHg or if there was asystole for >20 s. Fetuses that were able to complete the full 25-min period of occlusion showed no differences between sexes for any cardiovascular responses. Similar numbers of occlusions were stopped early in males (mean: 21 min, n = 16) and females (mean: 23 min, n = 16); however, they showed different responses. Short-occlusion males (n = 16) showed a slower initial fall in femoral vascular conductance, followed by greater bradycardia, hypotension, and associated organ hypoperfusion compared with full-occlusion fetuses. In contrast, short-occlusion females (n = 16) showed a significantly more rapid early increase in femoral vascular conductance than the full-occlusion fetuses, followed by worsening of bradycardia and hypotension that was intermediate to the full-occlusion fetuses and short-occlusion males. Among all fetuses, MAP at 15 min of occlusion, corresponding with the time of the maximal rate of fall, was correlated with postmortem weight in males (R(2) = 0.07) but not females. In conclusion, male and female fetuses showed remarkably similar chemoreflex and hemodynamic responses to severe asphyxia, but some males did show impaired hemodynamic adaptation within the normal weight range.     

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.     

Flight activity and oviposition characteristics of the seasonal form of a migrant skipper,Parnara guttata guttata (Lepidoptera: Hesperiidae)

The flight activity of adults of a migrant skipper (Parnara guttata guttata) was measured on the day of emergence, and at 4 and 7 days after emergence by a flight mill and the activity was compared among the three seasonal forms which emerged under 12, 14 and 16 hr photoperiods. The flight activity of adults reared under 12 and 14 hrs was higher than that under 16 hr especially on the day of emergence. The flight activity of males under 12 and 14 hrs was higher on the day of emergence and decreased at 4 and 7 days. The activity of males under 16 hr was maintained at a low and constant level. The activity of females under 12 and 14 hrs increased or was constant on the days after the emergence, on the other hand, the activity of females under 16 hr was highest at 4 days after the emergence. The wing size did not affect the flight activity. The oviposition characteristics of females reared under the three photoperiods was compared. The female under 14 hr has a longer preoviposition period and lesser fecundity than that under 16 hr. The female under 12 hr has intermediate values of the former two. The size of eggs laid by females under 12 hr was also intermediate.     

Differentiation of the müllerian (paramesonephric) duct epithelium in the rat

Differentiation of the Müllerian duct epithelium was studied in 15- to 21-day female rat foetuses. The proximal segment of the Müllerian duct is formed by the 15th day; it runs parallel to the Wolffian duct and the two are wrapped in a common basement membrane. On the 16th day the genital ducts are clearly separate; the Müllerian duct has a slit-like lumen and is lined with simple columnar epithelium. Throughout the whole of the given period the epithelium retains a relatively indifferent appearance. Characteristic findings from the 18th day include the apical migration of centrioles and the formation of solitary cilia.     

Experimental manipulation of sexual differentiation in wallaby pouch young treated with exogenous steroids

We have investigated the effects of androgen or oestrogen treatment of female or male tammar wallabies from the day of birth, when the gonads are histologically undifferentiated, to day 25 of pouch life, when the gonads and the Wolffian and Müllerian ducts have differentiated and the testes have migrated through the inguinal canal. Female tammars treated with testosterone propionate (24-50 mg kg-1 day-1) orally for 25 days had enlarged Wolffian and Müllerian ducts. Mammary and pouch development, however, was indistinguishable from that of control females. The treatment had no apparent effect on ovarian development, or on ovarian position in the abdomen. The phallus of males and females was similar in size, and neither experimental treatment had a significant effect on its size at day 25. Male tammars treated with oestradiol benzoate (1.2-2.5 mg kg-1 day-1) orally for 25 days had gross hypertrophy of the urogenital sinus. Testicular morphology was abnormal; many of the germ cells appeared necrotic, the seminiferous tubules were of reduced diameter, and there were few Leydig cells and increased amounts of fibrous tissue between the tubules. The cortex of these gonads contained some areas which had an ovarian appearance, lacking tubules and containing numerous germ cells. The Müllerian ducts of control males had regressed, but this was prevented by oestrogen treatment, suggesting an inhibition of either Müllerian Inhibiting Substance (MIS) production or its action. Normal testicular migration was inhibited in treated males; the testes remained high in the abdomen, similar in position to the ovaries of control females, whilst control males all had testes in the inguinal region. The gubernaculum and processus vaginalis of control males extended into the scrotum, but in treated males they terminated outside it. Oestrogen treatment had no effect on the size of the scrotum and did not induce mammary or pouch development. These experiments show that marsupials, like eutherians, have a dual hormonal control of Wolffian and Müllerian development. By contrast, the initial development of the mammary glands, pouch, gubernaculum and scrotum does not appear to be under hormonal control and is therefore likely to be autonomous and dependent on genotype.     

Virilization of the male pouch young of the tammar wallaby does not appear to be mediated by plasma testosterone or dihydrotestosterone.

Virilization of the male urogenital tract of all mammals, including marsupials, is mediated by androgenic hormones secreted by the testes. We have previously demonstrated profound sexual dimorphism in the concentrations of gonadal androgens in pouch young of the tammar wallaby Macropus eugenii during the interval when the urogenital sinus virilizes. To provide insight into the mechanisms by which androgens are transported from the testes to the target tissues, we measured testosterone and dihydrotestosterone in plasma pools from tammar pouch young from the day of birth to Day 150. Plasma testosterone levels were measurable (0.5-2 ng/ml) at all times studied, but there were no differences between males and females. These low concentrations of plasma testosterone appear to be derived from the adrenal glands and not the testes. Plasma dihydrotestosterone levels in plasma pools from these animals were also low and not sexually dimorphic. We conclude that virilization of the male urogenital tract cannot be explained by the usual transport of testosterone or dihydrotestosterone in plasma but may be mediated by the direct delivery of androgens to the urogenital tract via the Wolffian ducts. Alternatively, circulating prohormones may be converted to androgens in target tissues.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad‐intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad‐intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen‐responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor–immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28 ± 0.3 units) than in males (2.21 ± 0.2 units; p < .05). These sex differences in the number and staining intensity of androgen‐responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 359–370, 1999     

Immunofluorescent localization of tenascin during development of the mouse urogenital sinus: Possible involvement in genital duct morphogenesis

Tenascin is a compound of the mesenchymal extracellular matrix and has been proposed as a possible mediator in epithelial-mesenchymal interactions, because of its characteristic distribution in tissues during fetal development. In the present study, we have investigated by immunofluorescence the changes in the distribution of tenascin during development of the mouse urogenital sinus, a process in which tissue interactions were found to be essential. Tenascin first appears in dorsal mesenchyme on days 13-15 of gestation, coinciding with morphological changes of the epithelium. During male development, tenascin accumulates in the dorsal mesenchyme around the junction of Wolffian ducts, but not in the ventral mesenchyme, into which prostatic buds (prostate gland anlagen) project from the sinus epithelium. During female development, the mesenchyme that participates in the downgrowth of the vagina (derived from Müllerian ducts) stains intensively for tenascin. In both of these tenascin-positive areas, the epithelium undergoes conspicuous morphogenetic changes. The results suggest that mesenchymal tenascin could be involved in the epithelial morphogenesis of the sinus, especially in the morphogenesis of the genital ducts.     

Epithelial differentiation in the fetal rat colon: I. Plasma membrane phosphatase activities

During the last week of gestation of the fetal rat, the epithelium of the colon is rapidly remodeled. At 16 days a primitive stratified epithelium surrounds a small central lumen. Over the next 3 days, the main lumen extends narrow clefts down to the basal cell layer and small secondary lumina appear within the stratified epithelium between these clefts. At 19 and 20 days, secondary lumina enlarge but remain discrete; an infusion of cationic ferritin into the main lumen does not enter secondary lumina. During the 2 days prior to birth (21–22), the secondary lumina join the main lumen as superficial cells are sloughed, and the epithelium becomes simple columnar. Freeze-fracture replicas indicate that luminal and nonluminal membrane domains of epithelial cell plasma membranes are separated by continuous tight junctions throughout the conversion process. Cytochemical analysis of tissue slices from 16- to 22-day fetal colon demonstrated the appearance and segregation of two phosphatases on apical and basolateral membrane domains during epithelial conversion. Cysteine-sensitive pH 9.0 (alkaline) phosphatase activity was first detected along the luminal membranes of cells bordering both primary and secondary lumina at 18 days gestation and increased to a maximum at 20–21 days; weaker activity was present on basolateral membranes. Phosphatase activity at pH 8.0 also appeared at 18 days and increased thereafter, but was localized primarily on nonluminal membranes. At pH 8.0, reaction product appeared on both inner and outer sides of the membrane, and was only partially abolished by omission of K⁺ or addition of ouabain; thus the reaction may be only partially due to K⁺-dependent ATPase activity. Biochemical analysis of the cytochemical media confirmed the appearance of phosphatase activities at 18 days. Thus, plasma membrane phosphatase activities appear while the epithelium is still stratified, but are segregated to luminal and nonluminal membrane domains at the onset of activity. Segregation is maintained throughout the process of conversion of a simple columnar epithelium.     

Effects of gonadectomy and androgen treatment on aromatase activity in the fetal monkey brain

We studied the ability of neural tissues from fetal rhesus macaques to aromatize androgens to estrogens and assessed whether androgens are involved in the regulation of aromatase activity during development. Fetuses of both sexes, obtained on approximately Day 100 of gestation, were gonadectomized and immediately given intraabdominal Silastic capsules containing dihydrotestosterone. Sham-gonadectomized (intact) and gonadectomized, sham-implanted groups were also studied. Three weeks after the initial operation, the fetuses were delivered by cesarean section. To examine the developmental pattern of aromatase activity in the brain, fetuses were also delivered at two earlier times in gestation (at approximately 50 days and approximately 80 days). Whole-homogenates of preoptic area plus anterior hypothalamus (POA), hypothalamus (HT), amygdala (AMYG), and cerebral cortex (CTX) were incubated for 1 h in a phosphate buffer with saturating concentrations of [1 beta-3H] androstenedione. The amount of 3H2O formed was used as an index to estimate aromatase activity. The aromatase reaction exhibited Michaelis-Menten kinetics with an apparent Km of approximately 0.03 microM in all tissues from 120-day-old fetuses. Activity measured with the 3H2O assay closely corresponded with levels determined by product isolation. The highest levels of aromatase activity were found in the POA. Neither gonadectomy nor treatment with androgen affected aromatase in fetal tissue; activities in males were significantly greater than in females for the AMYG and CTX, but not for the POA or HT. The levels of aromatase activity detected in the HT and CTX of both sexes on Days 50 and 80 of gestation were at least ten times greater than the levels measured in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and accumulation of fibronectin in rabbit fetal lung tissue

An interaction between mesenchyme and epithelium is required for the normal differentiation of fetal lung tissue. This morphogenic interaction may be mediated, in part, by changes in the composition and/or structure of the extracellular matrix. Therefore, we characterized the localization and accumulation of fibronectin, an extracellular-matrix component, during several stages of lung development in the rabbit fetus in vivo as well as in day-21 rabbit fetal lung explants maintained in vitro. Fibronectin was detected immunocytochemically in the basement-membrane zone beneath the epithelial ducts in lung tissue obtained from rabbit fetuses at 19 and 21 days of gestation. In fetal lung tissue obtained at these early stages of lung development, mesenchymal cells were stained only at their periphery. Immunostaining for connective-tissue fibronectin increased greatly between days 24 and 31 of gestation. A similar increase in the intensity of immunostaining for connective-tissue fibronectin was observed in rabbit fetal lung explants that had been maintained in vitro for 7 days. The concentration of fibronectin in fetal lung tissue obtained at different days of gestation was determined using a specific enzyme-linked immunoadsorbent assay (ELISA) and was found to increase from 1.7 ng/micrograms protein in fetal lung tissue obtained at day 19 of gestation to 7.3 ng/micrograms protein in fetal lung tissue obtained at day 24 of gestation. The levels of fetal lung fibronectin then remained relatively constant through to day 31 of gestation. A similar increase in fibronectin concentration was observed in day-21 fetal lung explants maintained in vitro for 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in interstitial collagens of fetal rat genital ducts

The distribution of interstitial collagen types I and III was studied by immunocytochemistry in the mesenchyme of progressing and regressing mesonephric and paramesonephric ducts of male and female rat fetuses from the age of 15 days until birth. Immunocytochemistry revealed a collagen-poor mesenchymal area around the genital ducts and in continuation with the coelomic epithelium on the lateral edge of the mesonephric ridge of 15-day-old fetuses. Ultrastructurally, collagen fibrils were accumulated along the continuous lamina densa of the mesonephric ducts, whereas they were absent on the medial side of the male and female paramesonephric ducts. In males, the amount of collagen fibrils increased with the histological maturation of the mesenchyme around the mesonephric duct, whereas around the regressing paramesonephric duct collagens disappeared from the basement membrane region and the surrounding mesenchyme of the 16-day-old male duct. After the completion of the paramesonephric regression, the mesenchyme acquired a uniformly collagen containing interstitial matrix. In females, the collagens increased in the mesenchyme around the progressing paramesonephric duct, and the original site of the regressing mesonephric duct became occupied with a collagen-containing mesenchyme by the age of 19 days. The results suggest a close structural linkage between the mesonephric duct and the established early paramesonephric duct. The differences in the developmental maturation of the periductal mesenchyme and the observed changes in the composition of the interstitial matrix probably reflect the functional differences in the regulatory factors acting on the progression and regression of the male and female genital ducts.