Urogenital Tissues Research Articles

The globoseries glycosphingolipid sialosyl galactosyl globoside is found in urinary tract tissues and is a preferred binding receptor In vitro for uropathogenic Escherichia coli expressing pap-encoded adhesins.

Women with a history of recurrent Escherichia coli urinary tract infections (UTIs) are significantly more likely to be nonsecretors of blood group antigens than are women without such a history, and vaginal epithelial cells (VEC) from women who are nonsecretors show enhanced adherence of uropathogenic E. coli isolates compared with cells from secretors. We previously extracted glycosphingolipids (GSLs) from native VEC and determined that nonsecretors (but not secretors) selectively express two extended globoseries GSLs, sialosyl galactosyl globoside (SGG) and disialosyl galactosyl globoside (DSGG), which specifically bound uropathogenic E. coli R45 expressing a P adhesin. In this study, we demonstrated, by purifying the compounds from this source, that SGG and DSGG are expressed in human kidney tissue. We also demonstrated that SGG and DSGG isolated from human kidneys bind uropathogenic E. coli isolates expressing each of the three classes of pap-encoded adhesins, including cloned isolates expressing PapG from J96, PrsG from J96, and PapG from IA2, and the wild-type isolates IA2 and R45. We metabolically 35S labeled these five E. coli isolates and measured their relative binding affinities to serial dilutions of SGG and DSGG as well as to globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4), two other globoseries GSLs present in urogenital tissues. Each of the five E. coli isolates bound to SGG with the highest apparent avidity compared with their binding to DSGG, Gb3, and Gb4, and each isolate had a unique pattern of GSL binding affinity. These studies further suggest that SGG likely plays an important role in the pathogenesis of UTI and that its presence may account for the increased binding of E. coli to uroepithelial cells from nonsecretors and for the increased susceptibility of nonsecretors to recurrent UTI.

Urogenital aging—A hidden problem

Urogenital problems in the elderly female population are experienced by one third of women from the age 50 years and onward. Symptoms from the lower urinary tract include incontinence, urethritis, and recurrent urinary tract infections. Atrophic changes within the bladder neck and urethra could be corrected by estrogen administration even at doses so low that endometrial proliferation is avoided. Hence such estrogens could be given without progestogen comedication. Control of micturition is a complex process of which estrogen deficiency is only one of several factors. The aging process with subsequent changes in membrane permeability, neuromuscular function, and collagen synthesis contributes to the local problems of control of micturition. In addition, the central control may also be affected by degenerative changes of the nervous system. Vaginal symptoms comprise dryness of vagina, dyspareunia, and recurrent vaginitis often followed by a fowl odor and discharge. The microflora with lactobacilli and low pH as seen in fertile women is gradually replaced by a mixed germ flora including several of the pathogenic organisms common in urinary tract infections. Vaginal pH increases from around 4 to between 6 and 7. It is a puzzling fact that the urogenital tissues seem to be more "sensitive" to estrogens than other tissues. Conformational changes of the estrogen receptor(s) brought about by the local cytokine milieu is one possibility to explain the situation. The systemic absorption of low-dose estrogen preparations is dependent on the status of the vaginal mucosa. Absorption is high when the vaginal mucosa is atrophic and gradually decreases (but not to zero) as the vaginal mucosa matures under estrogen influence. (Am J Obstet Gynecol 1998;178:S245-9.)

In vivo effects of activated H‐ras oncogene expressed in the liver and in urogenital tissues

In vivo effects of activated H‐ras oncogene expressed in the liver and in urogenital tissues

In vivo effects of activated H‐ras oncogene expressed in the liver and in urogenital tissues

In vivo effects of activated H‐ras oncogene expressed in the liver and in urogenital tissues

In vivo effects of activated H-ras oncogene expressed in the liver and in urogenital tissues.

Transgenic mouse technology provides a direct genetic approach to in vivo carcinogenesis. In order to determine the oncogenic potential of an activated ras gene in liver, kidney and intestine, we created transgenic mice expressing the human H-ras oncogene under control of the L-type pyruvate-kinase gene. This gene is expressed in hepatocytes, enterocytes, proximal tubular cells of the kidney and endocrine pancreatic cells. Depending on lines, we observed hepatocarcinoma, polycystic kidney disease and an unexpected epididymis hyperplasia. These transgenic mice are an interesting model of polycystic kidney disease, and complete our study of the tissue-specificity of oncogene action.

In vivo effects of activated H-ras oncogene expressed in the liver and in urogenital tissues

In vivo effects of activated H-ras oncogene expressed in the liver and in urogenital tissues

CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae.

Is Kaposi's Sarcoma–Associated Herpesvirus Ubiquitous in Urogenital and Prostate Tissues?

AbstractControversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Is Kaposi's Sarcoma–Associated Herpesvirus Ubiquitous in Urogenital and Prostate Tissues?

Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Androgen metabolism in the prostate of the finasteride-treated, adult rat: a possible explanation for the differential action of testosterone and 5 alpha-dihydrotestosterone during development of the male urogenital tract.

Previous work has clearly demonstrated that inhibition of 5 alpha-dihydrotestosterone (DHT) formation in vivo is not as effective as total androgen ablation (castration) in causing involution of the prostate. It is likely that this is due to the fact that testosterone is partially effective in maintaining androgen action. To provide insight into this observation, the androgenic metabolites of testosterone, androstenedione, and 5 alpha-DHT, were measured in prostate tissue and in blood of 5 alpha-reductase inhibitor (finasteride)-treated adult male rats. Finasteride treatment caused a significant decrease in prostatic DHT levels and a profound increase in prostatic testosterone and androstenedione levels. Similarly, circulating DHT levels were decreased in finasteride-treated rats (0.02 ng/ml compared with 0.05 ng/ml seen in control rats); and circulating androstenedione and testosterone levels were significantly elevated in finasteride-treated animals compared with controls. The in vitro effects of finasteride were assessed on the metabolism of [3H]testosterone in a tissue-slice assays. In the prostate, the inhibition of 5 alpha-reductase activity resulted not only in the decreased formation of 5 alpha-reduced metabolites (primarily DHT and 5 alpha-androstanedione), but also an increase in the 17-oxo metabolite androstenedione. In contrast, the tissues derived from the embryonic wolffian duct (seminal vesicle and epididymis) formed relatively low amounts of 17-keto steroids. Because DHT is a high affinity ligand for the androgen receptor and androstenedione shows very little, if any, affinity for the receptor, these studies suggest that 5 alpha-reduction of testosterone may be a mechanism to amplify androgen action in urogenital tissues such as the prostate by preventing catabolism of testosterone to the inactive androgen, androstenedione, at the site of hormone action.

Effects of N-, P- and Q-type neuronal calcium channel antagonists on mammalian peripheral neurotransmission.

1. The effects of N-, P- and Q-type neuronal voltage-operated calcium (Ca2+) channel antagonists on neurotransmission were determined in a range of cardiovascular and urogenital tissues, as well as the diaphragm, isolated from rat or mouse. 2. The pharmacological tools chosen were omega-conotoxin GVIA (CTX GVIA), a selective N-type Ca2+ channel antagonist, the P-type channel blocker (< or = 100 nM) omega-agatoxin IVA (AGA IVA) and omega-conotoxin MVIIC (CTX MVIIC), a non-selective antagonist of N-, P- and Q-type channels. The effects of these antagonists on nerve-mediated responses were assessed in right atria, vasa deferentia, phrenic nerve-hemidiaphragms and small mesenteric arteries. 3. Rat mesenteric artery contractile responses to perivascular nerve stimulation were concentration-dependently inhibited by CTX GVIA (1-10 nM); inhibition was 92% with 10 nM. CTX MVIIC was > 100 fold less potent and only caused an inhibition of 46% at the highest concentration (1000 nM). AGA IVA (100 nM) had no effect. 4. In rat vas deferens stimulated at 0.05 Hz, CTX GVIA (10 nM) completely inhibited the twitch response and CTX MVIIC, about 100 fold less potent, caused total inhibition at 1000 nM. AGA IVA did not affect the twitch. In rat preparations stimulated at 20 Hz, a CTX GVIA-resistant (< or = 1000 nM) twitch response of 25% was apparent which could be blocked by 1000 nM AGA IVA or CTX MVIIC. In mouse vas deferens (20 Hz stimulation), CTX GVIA 10 nM caused an 87% inhibition of the twitch, the remainder being resistant to CTX GVIA, 100 nM. CTX MVIIC was only 10 fold less potent than CTX GVIA and completely inhibited the response at 1000 nM. AGA IVA (100 nM) inhibited the twitch by 55%. 5. The twitch response of the mouse phrenic nerve-hemidiaphragm was concentration-dependently inhibited by AGA IVA (1-100 nM); inhibition was 92% at 100 nM. CTX MVIIC was about 10 fold less potent than AGA IVA with an inhibition of 80% at 1000 nM. CTX GVIA was without effect. In the rat diaphragm preparation, AGA IVA (< or = 100 nM) and CTX GVIA (< or = 1000 nM) had little effect on the twitch response. CTX MVIIC (1000 nM) inhibited the twitch by 57%. 6. In rat and mouse right atria, sympathetic responses were concentration-dependently inhibited by CTX GVIA with almost complete block at 10-100 nM. CTX MVIIC was 100 fold less potent and caused complete inhibition at 1000 nM in the mouse preparation. AGA IVA did not affect atrial sympathetic responses. 7. These data suggest that N-type Ca2+ channels predominate in the control of sympathetic transmission in the mesenteric artery, vas deferens and right atrium. In the mouse vas deferens (and rat tissue at high stimulus frequency), P- and Q-type channels also mediate Ca2+ influx. P- and Q-type Ca2+ channels control neurosecretion at the motor endplate, with no role for N type channels.

Characterization of transferrin binding proteins 1 and 2 in invasive type b and nontypeable strains of Haemophilus influenzae.

Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa.

Cloning and characterization of a second AP-2 transcription factor: AP-2β

AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.

Cloning and partial sequence of the rabbit androgen receptor: expression in fetal urogenital tissues.

Genetic studies indicate that the actions of testosterone and dihydrotestosterone are mediated by a single androgen receptor (AR). The basis for the differential effects of these hormones during development is not known. To address this problem, we have turned to the rabbit, a species in which the endocrine and metabolic events in male sexual differentiation have been studied in detail. As a first step, we have cloned a partial cDNA encoding the rabbit prostate AR and have analyzed the expression of the cDNA in tissues of rabbit embryos at the time during which male phenotypic differentiation takes place. The coding sequence of the rabbit AR cDNA reveals a high degree of conservation with the sequences of the human, mouse, and rat ARs. By Northern analysis the principal transcript expressed in the mesonephros and the anlage of the external genitalia on gestation day 18 appeared to be identical to the mRNA expressed in adult prostate and epididymis.

Long-term gonadotropin-releasing hormone agonist therapy: the evolving issue of steroidal "add-back" paradigms.

The introduction of steroid "add-back" regimen draws on the recognition that several clinical entities targeted for treatment with GnRHa are not "six-month diseases". Included under this heading are individuals suffering from symptomatic endometriosis (not desirous of pregnancy), uterine fibroids (ineligible or disinterested in definitive surgical therapy), ovarian hyperandrogenism, premenstrual syndrome, menopausal transition, or dysfunctional uterine bleeding. A six month course of therapy with a GnRHa does not adversely affect lipoprotein economy and therefore presumably the corresponding cardiovascular risk. A six month course of GnRHa therapy appears to be associated with a substantial decrease (of up to 8.2%) in lumbar bone density, a phenomenon which may not be entirely reversible six months after discontinuation of therapy. In principle, steroid "add-back" therapy should diminish some or all of the side effects associated with GnRHa therapy, may provide a medical treatment option for patients representing a high surgical risk, and may delay surgical intervention if desired. On the other hand, a steroid "add-back" therapy may delay tissue diagnosis, be associated with a substantial cost as well as with the need in parenteral route of administration. Norethindrone-only (but not medroxyprogesterone acetate-only) "add-back" regimens have proved promising in the context of endometriosis. Non-concurrent estrogen/progestin "add-back" regimens proved promising in the context of uterine fibroids. Substantial additional studies would have to be carried out to validate the utility of steroid "add-back" regimens. Special emphasis will have to be placed on the evaluation of long-term utility with an eye towards assessing clinical efficacy, impact on lipoprotein economy, impact on bone density, impact on urogenital tissues, and impact on the hot flash. The concurrent or non-concurrent use of non-steroid "add-back" regimen will also most likely constitute a major component of future studies.

Changes in the density of nuclear estrogen receptors in urogenital tissue during pseudopregnancy in the rabbit

To elucidate the possible role of progesterone in pregnancy-induced urinary incontinence, the concentration of nuclear estrogen receptors (E2R) was measured in the urogenital tissues of non-pregnant (controls) and on days 6, 12 and 18 of pseudopregnancy in the rabbit. Whereas in the uterus E2R levels decreased by at least five-fold by day 6 of pseudopregnancy, changed little through day 12 and increased again at day 18, in the vagina and urethra only a small and insignificant decrease and increase occurred at days 6–12 and day 18, respectively. The receptor affinity, as judged by the computed KD values, did not change significantly in any of the preparation during pseudopregnancy. Similar changes in the E2R density were observed in rabbits treated with progesterone. These data provide further and concrete evidence for the tissue specificity in the downregulation of E2R by progesterone.

The androgen receptor of the urogenital tract of the fetal rat is regulated by androgen

To provide insight into androgen-mediated virilization, we measured the androgen receptor in tissues of male and female rat fetuses prior to and during the period of phenotypic sex differentiation. Western immunoblotting was performed utilizing an antibody directed against the 21 amino-terminal segment of the androgen receptor. In immunoblots prepared from urogenital tract tissues of day 17 male and female fetal rats, this antibody specifically recognizes a 11 OK protein band characteristic of the androgen receptor. Androgen receptor levels were low to undetectable in a variety of non-urogenital tract tissues. After day 18 of fetal development, the amount of androgen receptor decreased in female urogenital tissues, and by day 22 the amount of immunoreactive androgen receptor was higher in the male urogenital sinus and tubercle than in the corresponding tissues of the female. Administration of 5α-dihydrotestosterone to pregnant rats at a dose of 50 mg/kg body weight per day from day 12 to day 22 caused an increase in immunoreactive androgen receptor in the female urogenital sinus and tubercle to levels approaching those in male tissues. Administration of the androgen antagonist flutamide (100 mg/kg body weight per day) during the same interval caused a reduction in androgen receptor level in the urogenital sinus and tubercle of the male. These findings suggest that androgens modulate the amount of androgen receptor in the embryonic urogenital tract either by inducing the proliferation of androgen-responsive cells or by increasing androgen receptor levels in individual cells.

Isolation and biological activity of aspidospermine and quebrachamine from an Aspidosperma tree source.

The indolealkaloids aspidospermine and quebrachamine have been isolated in crystalline form by a relatively rapid fractionation from the extract of a powdered material designated "Quebracho" derived from an Aspidosperma tree species. We present a novel isocratic LC method that provides baseline resolution of these two compounds and of the structurally related yohimbine in less than 15 min. Gas chromatography-mass spectrometry was employed to identify these compounds as well as several minor derivatives of aspidospermine during and after the purification process. Aspidospermine and quebrachamine like yohimbine have been found to possess adrenergic blocking activities for a variety of urogenital tissues.

Uptake of 3H‐estradiol by embryonic crocodile gonads during the period of sexual differentiation

AbstractEgg incubation temperature controls sexual differentiation of the gonads in the saltwater crocodile, Crocodylus porosus. Embryos incubated at 30°C develop ovaries and hatch as females while most embryos incubated at 32–32.5°C develop testes and hatch as males. Several lines of evidence suggest that estrogen is involved in gonadal sex differentiation in crocodilians and other reptiles with temperature‐dependent sex determination (TSD). In vivo autoradiography was used here to identify sites of 3H‐estradiol uptake in the urogenital tissues of C. porosus embryos incubated at 30°C (female‐producing) and at 32.5°C (predominantly male‐producing). During the period of gonadal differentiation (developmental stage 23), 3H‐estradiol was specifically concentrated in the gonads of presumptive females at 30°C and presumptive males at 32.5°C. Binding was localised primarily in the medulla of the gonad in both sexes, with little or no uptake in the cortex. No uptake was detected in the adjoining adrenal glands, at either temperature, although some uptake was observed in the mesonephros at the female temperature (30°C). No specific binding of estradiol by the gonads was found prior to (stage 19) or after (stage 28) the period of gonadal differentiation. These results are consistent with the hypothesis that estrogen binding sites are present in the gonads of both sexes during a specific period of embryogenesis, which coincides with the time of gonadal sex differentiation. Furthermore, these observations suggest that the role of estrogen in TSD is not mediated through differential uptake of the steroid by presumptive ovaries and testes. It is therefore hypothesised that estrogen synthesis is temperature‐sensitive, rather than estrogen binding by the gonad. © 1994 Wiley‐Liss, Inc.

Studies on sex determination in the American alligator alligator mississippiensis

AbstractEstradiol‐17β was measured in the plasma and urogenital tissues of male and female alligator embryos. Hormone levels were generally very low and there was no significant differences between the sexes. There were no significant changes in estrogen levels during the period of gonadal differentiation in either sex. Alligator embryos incubated at male producing temperatures were feminized by small doses of estrogen applied to the egg shell. The antiestrogen, tamoxifen, masculinizes turtle and bird embryos, but, paradoxically, feminized alligator embryos at male producing temperatures. The contraceptive steroid, norethindrone, a progestin that is claimed to block estrogen synthesis, is the most potent estrogenic steroid tested on alligator embryos. A single dose of norethindrone applied to the egg shell caused massive hypertrophy of the Müllerian duct and feminized embryos at male producing temperatures. The androgen, dihydrotestosterone, had no detectable effect on male or female embryos at the doses tested. Undifferentiated urogenital tracts of embryos were cultured at 30 and 33°C in the presence of steroids, tamoxifen, or antibodies to steroids. None of the treatments had any effect on tissue differentiation. Tissues survived for up to 6 weeks, but there was no evidence of gonadal differential in vitro. © 1994 Wiley‐Liss, Inc.