Fetal Reproductive Tract Research Articles

Molecular pathways in placental-fetal development and disruption

The first trimester of pregnancy ranks high in priority when minimizing harmful exposures, given the wide-ranging types of organogenesis occurring between 4- and 12-weeks’ gestation. One way to quantify potential harm to the fetus in the first trimester is to measure a corollary effect on the placenta. Placental biomarkers are widely present in maternal circulation, cord blood, and placental tissue biopsied at birth or at the time of pregnancy termination. Here we evaluate ten diverse pathways involving molecules expressed in the first trimester human placenta based on their relevance to normal fetal development and to the hypothesis of placental-fetal endocrine disruption (perturbation in development that results in abnormal endocrine function in the offspring), namely: human chorionic gonadotropin (hCG), thyroid hormone regulation, peroxisome proliferator activated receptor protein gamma (PPARγ), leptin, transforming growth factor beta, epiregulin, growth differentiation factor 15, small nucleolar RNAs, serotonin, and vitamin D. Some of these are well-established as biomarkers of placental-fetal endocrine disruption, while others are not well studied and were selected based on discovery analyses of the placental transcriptome. A literature search on these biomarkers summarizes evidence of placenta-specific production and regulation of each biomarker, and their role in fetal reproductive tract, brain, and other specific domains of fetal development. In this review, we extend the theory of fetal programming to placental-fetal programming.

Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Ex vivo development of the entire mouse fetal reproductive tract by using microdissection and membrane-based organ culture techniques

Ex vivo explant culture is an appealing alternative to in vivo studies on fetal reproductive organ development. There is extensive literature on ex vivo methods of growing the fetal gonad. However, a method for culturing the whole fetal reproductive tract that has a different shape and size has not been documented. Here, with careful dissection and proper tissue orientation, we successfully cultured the entire bicornuate reproductive tracts from mouse embryos of both sexes on the Transwell insert membrane. The cultured reproductive tract system undergoes sexually dimorphic establishment and region-specific morphogenesis comparable to in vivo development of their counterparts. To test this culture method's applications, we used chemical treatment (dihydrotestosterone and BMS 564929) and genetic cellular ablation mouse model (Gli1-CreER; Rosa-DTA) to investigate the roles of androgen signaling and Gli1+ mesenchyme in Wolffian duct development. Dihydrotestosterone and BMS 564929 promoted the ectopic maintenance of Wolffian ducts in cultured XX tissues. The efficient and specific elimination of Gli1+ mesenchyme was successfully achieved in the cultured tissues, resulting in defective coiling of Wolffian ducts. These results demonstrate the amenability of this organ culture method for chemical and genetic manipulations that are otherwise difficult to study in vivo. Taken together, the establishment of this organ culture method provides a valuable tool complementary to in vivo studies for understanding fetal reproductive tract development in mice.

Insight into a first trimester human placental mechanism of fetal endocrine disruption

Human chorionic gonadotropin (hCG) is a placental hormone produced during pregnancy which regulates in part the development of fetal reproductive tract. hCG is thought to mediate effects of prenatal phthalates on the fetus. We have shown previously that phthalate-hCG and hCG-neonatal genitalia associations are both sex-specific. The aim was to directly measure, within the relevant critical window in the first trimester, modification of hCG regulation by fetal sex and by phthalate levels. Placental tissue samples (N=96, 45% male) were donated by women undergoing elective pregnancy termination at 2 clinics between gestational weeks 7 - 14. Tissues were dissected, snap frozen and stored at -80 C. mRNAs were measured by qPCR: PPARG, CGA, CGB and 2 housekeeping genes, RN18S and UBP1. PPARG encodes a transcription factor that regulates expression of hCG (encoded by CGA and CGB). Quantitation was done by relative standard curve. Sex was determined by measuring 2 Y-linked genes by PCR. All data were normalized, transformed, and analyzed using linear regression. Placental phthalate levels in the same sample set are being analyzed currently. There was no difference in the means of mRNAs between male and female fetuses. We measured a strong interaction of PPARG and fetal sex in the correlation of PPARG and CGA/CGB (p-interaction<0.005). In male placentas, one log10 unit in PPARG was associated with a 2.53 (95% CI 1.80, 3.26) log10 increase in CGA and CGB levels. In female placentas, the same association was null (β= 0.37 log10 units, 95% CI -0.32, 1.05). Associations with placental phthalate levels are forthcoming. These data offer novel insight into the basis for commonly reported sex-specific associations between prenatal phthalates and child health outcomes. hCG offers promise as a molecular biomarker that can conceptually and quantitatively connect sex-specific effects of prenatal exposures on placental mechanisms, and on fetal outcomes.

Ontogeny of estrogen receptors in human male and female fetal reproductive tracts

This paper reviews and provides new observations on the ontogeny of estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2) in developing human male and female internal and external genitalia. Included in this study are observations on the human fetal uterine tube, the uterotubal junction, uterus, cervix, vagina, penis and clitoris. We also summarize and report on the ontogeny of estrogen receptors in the human fetal prostate, prostatic urethra and epididymis. The ontogeny of ESR1 and ESR2, which spans from 8 to 21 weeks correlates well with the known “window of susceptibility” (7–15 weeks) for diethylstilbestrol (DES)-induced malformations of the human female reproductive tract as determined through examination of DES daughters exposed in utero to this potent estrogen. Our fairly complete mapping of the ontogeny of ESR1 and ESR2 in developing human male and female internal and external genitalia provides a mechanistic framework for further investigation of the role of estrogen in normal development and of abnormalities elicited by exogenous estrogens.

Interactive effects of nonylphenol and bisphenol A exposure with oxidative stress on fetal reproductive indices

Nonylphenol (NP) and/or bisphenol A (BPA) may have reproductive effects. Although the mechanisms of action remain unclear, steroid hormones biosynthesis, hypothalamus pituitary adrenal axis activity, oxidative stress, and crosstalk interaction of NP and BPA mixture and its pathways may play a contributory role. This cross-sectional study examined whether the interactive effects of NP/BPA and oxidative stress biomarkers played a role in reproductive indices (penis length and anogenital distance (AGD)) in 244 mother-fetus pairs. Four biomarkers of oxidative stress, (8-hydroxy-2′-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO2Gua), 8-iso-prostaglandin F2α (8-isoPF2α), and 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA)) were simultaneously analyzed using the high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method. No significant associations were found between reproductive indices and NP/BPA or oxidative stress biomarkers. Maternal exposure to a mixture of NP and BPA may enhance 8-OHdG. Interactive effects were found in the high 8-isoPF2α group, and prenatal NP exposure was inversely associated with penis length (β = −3.68 mm; p = 0.01). Similar results were noted among boys who were born to mothers in the high 8-isoPF2α group, in which BPA was inversely associated with penis length (β = −4.43 mm; p = 0.005). Our findings suggest important implications for prenatal exposure to oxidative stress, as evidenced by the 8-isoPF2α level. Thus, NP and BPA may interact to shape fetal reproductive tract development, particularly in boys. The interactive effects of NP/BPA, oxidative stress, and reproductive indices should be considered.

Tissue interactions and estrogenic response during human female fetal reproductive tract development

The role of tissue interactions was explored to determine whether epithelial differentiation within the developing human reproductive tract is induced and specified by mesenchyme in tissue recombinants composed of mouse vaginal mesenchyme + human uterine tubal epithelium (mVgM+hTubE). The tissue recombinants were grown in DES-treated ovariectomized athymic mice. After 2–4 weeks of in vivo growth, several vaginal specific features were expressed in the human tubal epithelium. The mesenchyme-induced effects included morphological change as well as expression of several immunohistochemical markers. Although the mesenchyme-induced shift in vaginal differentiation in the human tubal epithelium was not complete, the partial induction of vaginal markers in human tubal epithelium verifies the importance of mesenchymal-epithelial interactions in development of the human female reproductive tract.In a separate experiment, DES-induction of uterine epithelial progesterone receptor (PGR) and estrogen receptor 1 (ESR1) was explored in tissue recombinants composed of wild-type or Esr1KO mouse uterine mesenchyme + human fetal uterine epithelium (wt UtM+hUtE and Esr1KO UtM+hUtE). The rationale of this experiment was to determine whether DES-induction of PGR and ESR1 is mediated directly via epithelial ESR1 or indirectly (paracrine mechanism) via mesenchymal ESR1. DES-induction of uterine epithelial ESR1 and PGR in Esr1KO UtM+hUtE tissue recombinants (devoid of mesenchymal ESR1) formally eliminates the paracrine mechanism and demonstrates that DES induction of human uterine epithelial ESR1 and PGR is directly mediated via epithelial ESR1.

Response of xenografts of developing human female reproductive tracts to the synthetic estrogen, diethylstilbestrol

Human female fetal reproductive tracts 9.5–22 weeks of gestation were grown for 1 month in ovariectomized athymic adult female mouse hosts that were either untreated or treated continuously with diethylstilbestrol (DES) via subcutaneous pellet. Normal morphogenesis and normal patterns of differentiation marker expression (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19, ESR1, PGR, TP63, RUNX1, ISL1, HOXA11 and α-ACT2) were observed in xenografts grown in untreated hosts and mimicked observations of previously reported (Cunha et al., 2017) non-grafted specimens of comparable age. DES elicited several notable morphological affects: (a) induction of endometrial/cervical glands, (b) increased plication (folding) of tubal epithelium, (c) stratified squamous maturation of vaginal epithelium and (d) vaginal adenosis. DES also induced ESR1 in epithelia of the uterine corpus, cervix and globally induced PGR in most cells of the developing human female reproductive tract. Keratin expression (KRT6, KRT7, KRT8, KRT14 and KRT19) was minimally affected by DES. Simple columnar adenotic epithelium was devoid of TP63 and RUNX1, while DES-induced mature vaginal epithelium was positive for both transcription factors. Another striking effect of DES was observed in grafts of human uterine tube, in which DES perturbed smooth muscle patterning. These results define for the first time IHC protein markers of DES action on the developing human reproductive tract, which provide bio-endpoints of estrogen-induced teratogenesis in the developing human female reproductive tract for future testing of estrogenic endocrine disruptors.

Maternal inflammatory bowel disease and hypospadias in male offspring: a population-based study in Denmark

BackgroundThe occurrence of inflammatory bowel disease (IBD) and hypospadias has been concurrently increasing, possibly through shared environmental risk factors such as endocrine disrupting compounds. Also, maternal IBD may disturb the...

1161 Development of the fetus and fetal reproductive tract in gilts subjected to heat stress from week 4 to 8 of gestation

1161 Development of the fetus and fetal reproductive tract in gilts subjected to heat stress from week 4 to 8 of gestation

Endometriosis: a new cellular and molecular genetic approach for understanding the pathogenesis and evolutivity.

Endometriosis is a benign disease with high prevalence in women of reproductive age estimated between 10 and 15% and is associated with considerable morbidity. Its etiology and pathogenesis are controversial but it is believed to involve multiple genetic, environmental, immunological, angiogenic, and endocrine processes. Altered expressions of growth factors, cytokines, adhesion molecules, matrix metalloproteinases, and enzymes for estrogen synthesis and metabolism have been frequently observed in this condition. The possibility of genetic basis of endometriosis is demonstrated in studies of familial disease, in which the incidence of endometriosis is higher for first-degree relatives of probands as compared to controls. This review describes mainly the cellular, cytochemical, cytogenetic, and molecular genetic features of endometriotic lesions and cultured endometriotic cells. In attempts to identify candidate gene (s) involved in the pathogenesis of endometriosis, a tissue-based approaches including conventional cytogenetics (RHG-banding), loss of heterozygosity (LOH), and comparative genomic hybridization (CGH) were employed. In addition to the karyotypic anomalies, consistent chromosome instability was confirmed by CGH and fluorescence in situ hybridization (FISH). The nature and significance of the molecular genetic aberrations in relation to the locations and function of oncogenes and tumor suppressor genes will be discussed. At last, a possible pathogenic role of embryonic duct remnants was observed in seven female fetal reproductive tract in endometriosis and may induce a discussion about the beginning of ovarian tumors and malignant proliferations.

Identification of Displaced Endometrial Glands and Embryonic Duct Remnants in Female Fetal Reproductive Tract: Possible Pathogenetic Role in Endometriotic and Pelvic Neoplastic Processes

Background: Recent findings strongly promoted the hypothesis that common pelvic gynecological diseases including endometriosis and ovarian neoplasia may develop de novo from ectopic endometrial-like glands and/or embryonic epithelial remnants. To verify the frequency, the anatomical localization and the phenotype of misplaced endometrial tissue along the fetal female reproductive tract, histological and immunohistochemical analyses of uteri, fallopian tubes, and uterosacral ligaments were performed. Methods: Reproductive organs were collected from seven female fetuses at autopsy, five of them from gestational ages between 18 and 26 weeks and two fetuses with gestational ages of 33 and 36 weeks deceased of placental anomalies. Serial sections from areas containing ectopic glands and embryonic duct residues were analyzed by histological and immunohistochemical procedures. Results: Numerous ectopic endometrial glands and stroma were detected in the myometrium in two fetuses with low levels of expression of estrogen receptor-alpha (ER-α) and progesterone receptors (PR). The embryonic ducts were localized in the uterine broad and ovarian ligaments and under the fallopian tube serosa in six fetuses. Low levels of steroid receptors expression were found in the embryonic residues, whereas the carcino-embryonic antigen (CEA) and the tumor marker Ca 125 were not detected. The embryonic residues stromal component strongly expressed the CD 10 and vimentin proteins. Conclusion: The anatomical and the immunohistochemical features of the ectopic organoid structures identified in fetal female reproductive tract suggest that endometriotic as well as neoplastic disease in adult women may develop on the basis of misplaced endometrial glands and/or embryonic cell remnants.

Early Expression of the Androgen Receptor in the Sertoli Cells of a Marsupial Coincides with Downregulation of Anti-Müllerian Hormone at the Time of Urogenital Virilization

Anti-Müllerian hormone (AMH), responsible for the regression of Müllerian ducts, is strongly expressed by eutherian fetal and postnatal Sertoli cells. Both AMH and testosterone levels are high during the period of fetal reproductive tract virilization which occurs largely in utero in eutherian mammals. Taking advantage of the fact that differentiation of the urogenital tract occurs after birth in marsupials, we studied the ontogeny and regulation of AMH in the tammar wallaby testis and related it to the expression of the androgen receptor in Sertoli cells. Testicular AMH expression was high between days 10–30 post partum, then fell to basal levels by day 60 and remained low until day 90, the oldest age examined. AMH expression was repressed by treatment of male pouch young with the potent androgen androstanediol. Thus, in the tammar, AMH expression decreases in response to androgen at the time of initial urogenital masculinization, in contrast to the situation in humans in which AMH is repressed by testosterone only at the time of puberty. The difference might be explained by the timing of androgen receptor expression which appears in tammar Sertoli cells at around day 40 of pouch life but only at a later developmental stage in eutherians.

Gene expression change in the Müllerian duct of the mouse fetus exposed to diethylstilbestrol in utero.

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 microg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.

“Natural” progesterone: information on fetal effects

A variety of progestational agents have been used therapeutically and evaluated for adverse effects over the last 50 years. However, progesterone itself has come into use as a therapeutic agent only recently with the development of an orally bioavailable "micronized" preparation. The current review examines progesterone adverse effects as identified in the larger literature on the toxicity of progestational agents and pharmacokinetics. Progesterone has cytoplasmic and membrane receptors in a variety of reproductive and nonreproductive tissues including the brain and is a potent inhibitor of GnRH. Limited information is available on progesterone receptors and actions in the fetus. Concern about exogenous progestagen effects on fetal reproductive tract development have led to considerable human research over the years, but this literature review demonstrates that contemporary developmental toxicology research on progesterone is lacking. Progesterone is a potent, multi-faceted endocrine agent with an expanding therapeutic profile and a minimal scientific database for evaluating safe use during pregnancy.

Immunohistochemical localization of cyclooxygenase-1 and cyclooxygenase-2 in the human fetal and adult male reproductive tracts.

The first rate-limiting step in the conversion of arachidonic acid to PGs is catalyzed by cyclooxygenase (Cox). Two isoforms of Cox have been identified, Cox-1 (constitutively expressed) and Cox-2 (inducible form), which are the products of two different genes. In this study we describe the immunohistochemical localization of Cox-1 and -2 in the human male fetal and adult reproductive tracts. There was no Cox-1 expression in fetal samples (prostate, seminal vesicles, or ejaculatory ducts), and only minimal expression in adult tissues. There was no expression of Cox-2 in the fetal prostate. In a prepubertal prostate there was some Cox-2 expression that localized exclusively to the smooth muscle cells of the transition zone. In adult hyperplastic prostates, Cox-2 was strongly expressed in smooth muscle cells, with no expression in the luminal epithelial cells. Cox-2 was strongly expressed in epithelial cells of both fetal and adult seminal vesicles and ejaculatory ducts. The Cox-2 staining intensity in the fetal ejaculatory ducts during various times of gestation correlated with previously reported testosterone production rates by the fetal testis. These data indicate that Cox-2 is the predominant isoform expressed in the fetal male reproductive tract, and its expression may be regulated by androgens. The distinct cell type-specific expression patterns of Cox-2 in the prostate (smooth muscle) vs. the seminal vesicles and ejaculatory ducts (epithelium) may reflect the different roles of PGs in these tissues.

Modulation of androgen receptor (AR)-mediated transcriptional activity by EGF in the developing mouse reproductive tract primary cells

Recently, a role for epidermal growth factor in male sexual differentiation was reported from different laboratories. We demonstrated that androgen receptor (AR) mediates the EGF-induced effects. The mechanism, by which EGF modulates AR mediated activity, is not known and the current studies were designed to investigate the role of AR. Using mesenchymal cell preparation from the 18-day fetal reproductive tract, first, we determined whether EGF induced sexual differentiation by enhancing AR gene expression. Thus, AR mRNA and AR protein levels were measured in response to EGF-treatment using RT-PCR based analysis of AR mRNA and Western blot analysis of AR protein level respectively. Both of these analysis detected presence of AR gene expression in this cell preparation, however no effect of EGF was observed in AR protein or AR mRNA expression. Next, we examined whether EGF enhanced AR mediated transcriptional activity in the developing tract. Using the mesenchymal cells, co-transfected with AR expression vector (pSV-AR) and androgen response element linked to luciferase reporter vector (pMAMneoLUC) we assessed AR-mediated transcriptional activity in response to EGF treatment in the presence and absence of testosterone. The results showed that androgen stimulated the luciferase activity in a dose dependent manner, as expected. EGF also enhanced such activity; however, the response was significantly lower than that by androgen. EGF, however, produced striking enhancement of the androgen-induced transcriptional activity when used with androgen. EGF and testosterone produced no stimulation of the luciferase activity either alone or in combination in the cells lacking AR expression vector, suggesting a role for AR in the effect of EGF and testosterone. Flutamide, an AR antagonist, also blocked the enhancement of luciferase activity induced by EGF and testosterone, further confirming the role of AR in the effect of EGF and testosterone. Thus, it appears that EGF-modulation of sexual differentiation involves enhancement of AR-mediated transcriptional activity and not enhancement of AR gene expression. Additionally, it appears that EGF modulates sexual differentiation in the presence of testosterone possibly by potentiating the testosterone-effect.

Cellular localization of immunoreactive epidermal growth factor during Wolffian duct differentiation of the fetal mouse.

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male reproductive tract differentiation of the fetal mouse. Expression of an EGF-like protein during Wolffian duct differentiation was indicated from the determinations by radioimmunoassay (RIA) and radioreceptor assay. To further characterize the protein and to assess its role in male sexual differentiation, expression of the protein has been analyzed by Western blot assay and its tissue-specific cellular expression has been determined by immunocytochemical assay in the present study. Western blot analysis of the 18-day fetal male reproductive tract detected an immunoreactive band of the predicted 6-kDa size. Immunocytochemical analysis also detected EGF-specific immunostaining in the Wolffian duct derivatives. At day 18 of gestation, the staining was localized predominantly in the epithelial nuclei of the Wolffian duct derivatives whereas at days 14 and 16 of gestation, the staining was equally distributed in the mesenchymal and epithelial sites of the Wolffian duct derivative. The intensity of the staining increased with progression of differentiation during the 14th-18th days of gestation. Prenatal exposure to the antiandrogen flutamide significantly reduced the immunostaining of the duct. Thus, a role for EGF in Wolffian duct differentiation is indicated.

Activation of androgen receptor in epidermal growth factor modulation of fetal mouse sexual differentiation

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male sexual differentiation. The mechanism by which EGF modulates male sexual differentiation has not been determined and investigation has been made to assess the role for androgen receptor (AR) in mediating the EGF-induced effect. We report that EGF, like androgen, stabilized the Wolffian duct in the 13-day female specimen, grown in organ culture. Anti-AR, flutamide and cyproterone acetate blocked the Wolffian duct-stabilizing effect of EGF. EGF also induced cell proliferation of the fetal reproductive tract in a dose-dependent manner and a combination of physiological dosages of EGF and androgen-induced cell proliferation synergistically, suggesting an interactive effect of these two drugs. Cyproterone acetate blocked both EGF-induced normal cell proliferation and the synergistic cell proliferation induced by combination of EGF and androgen suggesting a role of AR in the effects of EGF. The role of AR was further assessed by determining the effect of EGF on AR binding directly. It was shown that EGF stimulated androgen binding activity of the male fetal reproductive tract cells significantly by increasing the number of binding sites by 3-fold with slight decrease in binding affinity. Thus, it appears that AR plays a role in mediating EGF-modulation of sexual differentiation.

The role of growth hormone in fetal mouse reproductive tract differentiation.

Although GH plays a key role in postnatal growth and differentiation, its role in fetal differentiation is not clear at the present. The aim of the present study was to investigate whether GH plays a role in fetal sexual differentiation, and we used in vitro organ culture assay of sexual differentiation to determine this. The results showed that anti-rGH antibody blocked Wolffian duct differentiation specifically in the presence of fetal testes. Exogenous GH supplemented in the above experiment reversed the blocking effect of anti-GH. Among the other related products, insulin-like growth factor I was highly effective in reversing the anti-GH effect, insulin-like growth factor II was partially effective, but PRL was unable to reverse the anti-GH effect. GH itself was found to produce some masculinizing effect, as demonstrated by its ability to stabilize the Wolffian duct in female fetuses. The role of GH was further demonstrated by the observation that GH-immunoreactive material of the size of authentic GH was detected in the 18-day fetal reproductive tract, and the concentration of this material increased in response to progression of sexual differentiation. Determination of androgen-binding activity using Scatchard analysis on the cells isolated from the 18-day fetal reproductive tract indicated that androgen-binding activity increased after GH treatment of the cells. Thus, it may be concluded that GH influences male sexual differentiation and alters the androgen-binding activity of the fetal reproductive tract.