SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

In vitro androgenic induction of a major protein in epithelial cell subcultures from mouse vas deferens.

Pure epithelial cell cultures, obtained from primary culture of vas deferens tissue collected from 20- to 30-day-old mice, were amplified by subculturing the cells over 3T3 feeder layer in a serum-free defined medium. Adhesion and proliferation of epithelial cells did not require androgens, but a minimal concentration of 5.10(-7) M hydrocortisone. In that system, epithelial cells expressed cytokeratin but failed to produce the tissue specific mouse vas deferens protein (MVDP) in response to androgens. Various culture procedures and medium compositions were assayed for induction of MVDP expression. Culture onto microporous membrane inserts, which allow polarization of cells, is absolutely required for androgenic induction of MVDP. Androgen action did not require the presence of hydrocortisone, insulin, triiodothyronine, pituitary extracts, epidermal growth factor and acetylcholine. A minimal supplemented medium was then defined in which the expression of MVDP by epithelial cells in response to androgens was dose dependent. It has also been shown that this response at each concentration of dihydrotestosterone was heterogeneous at individual cell level. Highly reproducible results were obtained from epithelial cell cultures between 8th to 16th passages, showing that subcultured cells have maintained their ability to differentiate and express specialized functions.

Changes in the testosterone to dihydrotestosterone ratio in plasma and testes of maturing rabbits.

Testosterone (T) and dihydrostestosterone (DHT) were radioimmunologically assayed in the testes and plasma of rabbits at 1, 10, 20, 40, and 60 days of age and at 3, 4, 5, and 8 months. In the testis (nanograms per 100 mg testis) and plasma (picograms per ml), both hormones are low at birth, reach their maxima between 60--90 days, and then decline and remain low after 120 days. In the testes, the T:DHT ratio is very high from birth (4.2 +/- 0.4) to 60 days (8.8 +/- 1.1). A significant decrease (P less than 0.001) occurs between 60 (8.8 +/- 1.1) and 90 (1.2 +/- 0.1) days. The T:DHT ratio is always less than or equal to unity after 90 days. From 1--60 days, T is the dominant testicular hormone, while the levels of DHT are greater than or equal to those of T after 60 days. The mean value of the plasma T:DHT ratio is always greater than unity from birth (1.7 +/- 0.4) to adulthood (2.6 +/- 0.7). The dominant circulating hormone, at all ages, is T. Our results show that sexual maturation in rabbits is characterized by an inversion of the T:DHT ratio in the testes but not in the plasma.

Developmental patterns of the testicular response to experimental modifications of circulating androgen levels in the fetal rabbit.

The circulating level of free androgens in fetal and newborn rabbits was reduced by active immunization of mothers against testosterone (T) or was increased by injecting dihydrotestosterone (DHT) into the mothers. After immunization, about 100% of the circulating T and DHT in fetuses was bound. After maternal injection of DHT, the circulating level of this hormone in fetuses was increased 2- to 120-fold. The effects of these treatments were evaluated by determining testicular levels of T, a physiological index of circulating gonadotropin. From 20--23 days of gestation, testicular T content was modified neither by circulating antibodies nor by an increase of blood DHT. The same overloads of circulating DHT, which were ineffective between 20--23 days, significantly reduced testicular T content between 25--29 days. Testicular T content was significantly increased in newborns from immunized mothers. These results suggest that the appearance of the negative feedback action of circulating androgens takes place relatively late, at 24 or 25 days, after differentiation of the genital tract.

Levels of testosterone in the plasma, gonads, and adrenals during fetal development of the rabbit.

Testerone (T) concentrations in the plasma, gonads, and adrenals were measured by radioimmunoassay in 188 male and 160 female rabbit fetuses. Determinations were performed daily from the 20th to the 31st day of gestation and were correlated with the maternal plasma concentration of T. The T content of both testes remained relatively constant from the 20th (3040 pg) to the 26th day (3940 pg), and subsequently decreased until the 31st day (1630pg). the concentration of testicular T fluctuated only slightly from the 20th (1040 pg/mg) to the 23rd day (783 pg/mg), and thereafter decreased until the 31st day (138 pg/mg). The T levels in plasma of males (132-361 pg/ml) were significantly higher than those of females (21-116 pg/ml). Plasma T levels in males were relatively constant and did not exhibit any rise, which is similar to observations of the testis during differentiation of the genital tract. Testosterone concentrations were low in the adrenals (3.5-12.3 pg/mg) of both sexes and in the ovaries (1.5-20.4 pg/mg) of fetuses. These data provide the first evidence for testicular T secretion at the time of genital differentiation in the rabbit.

5'-flanking and intragenic sequences confer androgenic and developmental regulation of mouse aldose reductase-like gene in vas deferens and adrenal in transgenic mice.

The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.

Structure and sequence of a mouse gene encoding an androgen-regulated protein: a new member of the seminal vesicle secretory protein family.

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids), an androgen-dependent protein specifically expressed in the mouse seminal vesicle, was isolated and sequenced. A mouse genomic library constructed in the lambda EMBL12 vector was screened using a full length cDNA probe. One genomic clone was selected, 7.4 kb of which were shown to contain the whole MSVSP99 gene. The complete sequence of the MSVSP99 gene (1.7 kb), plus 0.8 and 0.3 kb of the 5' and 3' flanking regions respectively, has been determined. The gene is composed of four exons interrupted by three introns. The size range for the four exons is 47-217 bp, while that of introns is 87-615 bp. The transcription start site was identified as an adenine residue located 21 nucleotides upstream from the ATG start codon. Putative TATA and CAAT boxes were identified, along with a number of regions that shared homologies with known regulatory sequences. These included androgen-responsive elements located in the promoter as well as in the gene sequence. Sequence comparisons with other androgen-responsive genes showed strong homologies between the MSVSP99 gene and the seminal vesicle secretory protein (SVS) family genes (rat SVS II, IV, V and VI). Moreover, some regions were found to be conserved between the MSVSP99 gene and the human semenogelin I and II genes.

Structural and functional analysis of the promoter of a mouse gene encoding an androgen-regulated protein (MSVSP99).

MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is a member of the rat and mouse seminal vesicle secretory protein family. The gene encoding MSVSP99 is under androgenic control and we demonstrate here that this regulation involves a complex interplay of positive and negative regions. First, we show that the promoter region (-387/+16) sufficient to mediate a full androgen induction is a complex enhancer organized in two regulatory regions. These two regions are inactive individually and must act together to confer a 40-fold androgen induction to the MSVSP99 gene and androgen responsiveness is not only dependent on the presence of functional androgen response element (ARE) sequences but results from complex cooperations between ARE and non-ARE sequences forming an androgen response unit. Secondly, we characterized a new regulatory region (-824/-632) that decreases androgen-dependent transcriptional activity of the MSVSP99 promoter. This region, also able to repress the transcriptional activity of the heterologous thymidine kinase promoter, contains a functional promoter on the inverted strand (-826 to -387) and we identified a transcription initiation site located at position -639 with respect to the cap site of the MSVSP99 promoter. Sequence analysis of the flanking DNA also revealed that the MSVSP99 gene is surrounded by long interspersed repeated sequences called LINEs.

Lack of expression of the mRNA encoding a major protein of the mouse vas deferens after neonatal exposure to oestrogens.

We have previously characterized an androgen-inducible secretory protein from the mouse vas deferens (MVDP), and a cDNA to its mRNA has been obtained. This report describes altered MVDP gene expression after neonatal exposure to oestrogens. As shown by immunohistochemistry and Western blot analysis, MVDP was missing in the vas deferens from adult mice neonatally exposed to oestrogens. Northern blot analysis showed that the expression of MVDP mRNA was also suppressed. Exogenous testosterone was unable to stimulate MVDP production (either message or protein) in neonatally oestrogenized males. The results suggest that the alterations in gene expression in the oestrogen-exposed vas deferens reflect changes in the programme of differentiation of the organ itself.

Cloning and sequence analysis of a cDNA encoding an androgen-dependent mouse seminal vesicle secretory protein.

We report the cloning and sequencing of a new cDNA sequence encoding a protein from the mouse seminal vesicle. An open reading frame of 297 nucleotides encoded a protein of 99 amino acids with a calculated molecular mass of 11.454 kDa. The first 21 amino acids constituted a signal peptide followed by 78 amino acids encoding the secreted protein. The cDNA sequence comprised a 3' untranslated region of 226 bp and the polyadenylation signal AATAAA, 19 bp upstream from the poly(A)+ tail. A high degree of homology was found between this protein and members of the family of seminal vesicle secretory (SVS) proteins, especially rat SVS VI. Northern blot analysis indicated the presence of a 0.7 kb mRNA species in the mRNAs of seminal vesicle tissue. Castration resulted in a marked decrease in the level of the 0.7 kb mRNA encoding the protein, whereas administration of testosterone to castrated males restored the 0.7 kb mRNA.

Hormonal and developmental regulation of the mouse aldose reductase-like gene akr1b7 expression in Leydig cells.

The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.

Vas deferens epithelial cells in subculture: a model to study androgen regulation of gene expression.

The understanding of androgen-regulated gene expression requires a cell cultures system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24h of androgen induction. DEAE-dextran transfection conditions were defined using the MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT gene and transfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.

Radioimmunoassay of mouse vas deferens protein: developmental profile and hormonal regulation.

This study investigated the hormonal regulation of the developmental pattern of a major protein of the mouse vas deferens (MVDP) which represents 10% of the vas deferens protein content. Using a specific radioimmunoassay, MVDP was first detected in 10-day-old males and its concentration increased sharply between 10 and 30 days, reaching adult levels at 40 days. In adult males, MVDP disappeared after castration, and testosterone treatment for 15 days was necessary to reverse the effect of castration completely. In 10-day-old mice castrated at birth, MVDP levels were similar to those measured in controls of the same age. Testosterone administration from 1 to 10 days of age did not induce precocious accumulation of MVDP. These results suggested that the neonatal expression of MVDP is independent of androgens. From 10 to 30 days, the increase in MVDP levels was androgen-dependent as shown by the castration and injection experiments. In males castrated at birth, the androgen-induced accumulation of MVDP was greatly reduced in adulthood. This suggested that neonatal androgens are necessary to obtain full expression of the MVDP gene in adults.

Sex-related differences in renal size in mice: ontogeny and influence of neonatal androgens.

Kidneys of adult male mice are larger than those of females because of both cellular hyperplasia and hypertrophy. Administration of testosterone to adult female mice induced cellular hypertrophy but not hyperplasia, so that the weight of the kidney remained smaller than in male mice. The sexual dimorphism in kidney size is not congenital but programmed by neonatal endogenous androgens and expressed between 30 and 40 days of age. Treatment of newborn males with cyproterone acetate and of newborn females with testosterone induced female and male patterns of renal growth respectively. It appears that neonatal endogenous androgens are required to induce the characteristic cellular hyperplasia of the kidneys of male mice. Manipulation of androgen levels during neonatal and prepubertal life was found to affect the growth response of the kidney to androgens in adult male and female mice.

Sexual maturation in male mice treated with cyproterone acetate from birth to puberty.

Cyproterone acetate was administered every 2 days from 1 to 39 days of age to male mice which were killed 24 h or 20 days after the last injection. Cyproterone acetate caused a significant reduction in the relative weights of the epididymis, vas deferens, seminal vesicle and preputial gland, which was still evident at 60 days after birth. Testicular and epididymal androgens (testosterone and dihydrotestosterone) and circulating LH and FSH concentrations were equal to or higher than those of controls at 60 days. Cyproterone acetate did not inhibit spermatogenesis but all males were infertile. The results suggest that the peripheral effects of testosterone are necessary, during early stages of sexual maturation, in order to obtain subsequent full development of the accessory sex organs.

Pituitary and plasma levels of luteinizing hormone and follicle-stimulating hormone in male and female rabbit fetuses.

To study the ontogenesis of fetal pituitary gonadotrophin synthesis and release, LH and FSH were measured by radioimmunoassay in fetal rabbit pituitary glands and blood of both sexes from day 18 of gestation until birth. Results on levels of testicular and plasma testosterone were also included. Immunoreactive LH was first detected on day 19 in the pituitary gland and on day 20 in the plasma of fetuses of both sexes. Pituitary FSH was first measurable in both male and female fetuses at 24 days of gestation. Levels of FSH could not be detected in the blood of male fetuses at any time during gestation. In females, FSH could be measured in the circulation from day 27 of gestation until birth. These results show that (1) the ontogeny of pituitary LH and the onset of testosterone secretion are closely correlated and take place between 18 and 20 days of gestation just before the beginning of differentiation of the male genital tract, and (2) the highest concentrations of pituitary LH and FSH are observed, in both sexes, in late gestation after days 24-25.

Effects of dense housing on the growth of reproductive organs, plasma testosterone levels and fertility of male mice.

Male mice were raised in populations of two different sizes from birth to adulthood: six animals (three of each sex) or 30 animals (15 of each sex) being maintained in cages of the same dimensions. Animals were killed every 10 days from weaning to 60 days and at 90 days. Increased population size induced decreased body and seminal vesicle weights; testicular, pituitary and adrenal weights were little affected. Plasma testosterone levels were lowered by increasing population size over the period from weaning to 50 days but they were increased at 60 and 90 days. The age at which first fertile matings occurred was not affected. These results indicate that the endocrine function of the testis, as determined by the measures used here, was specifically affected by this differential housing.

Cyclic AMP regulates expression of the gene coding for a mouse vas deferens protein related to the aldo-keto reductase superfamily in human and murine adrenocortical cells.

Mouse vas deferens protein (MVDP) is a member of the aldo-keto reductase superfamily. The regulation of MVDP gene expression by activators of the protein kinase A signalling pathway was investigated in human (H295-R) and murine (Y1) adrenocortical carcinoma cells. Immunoblotting with polyclonal antibodies showed that MVDP is expressed in adrenal glands from mouse, rat, rabbit and guinea-pig, probably under the control of ACTH. In both adrenocortical cell lines used, MVDP is constitutively synthesized and its accumulation is increased by treatment with cAMP or forskolin. MVDP mRNA steady-state levels were up-regulated by forskolin in adrenocortical cells by a process that does not require de novo protein synthesis. The results suggest that cAMP is at least one of the key regulators of adrenal MVDP expression and that this effect is direct.

Permanent changes in the functional development of accessory sex organs and in fertility in male mice after neonatal exposure to cyproterone acetate.

Male mice were injected daily with cyproterone acetate for 10 consecutive days during one of the four following periods: 1-10 days, 11-20 days, 21-30 days or 31-40 days. At all stages studied cyproterone acetate caused a significant reduction in the relative weights of epididymis, vas deferens, preputial gland and seminal vesicle in males killed 24 h after the last injection; the androgen content (testosterone + dihydrotestosterone) of the accessory sex organs was also reduced but the differences were not always significant. Cyproterone acetate treatment from 1 to 10 days resulted in a definitive reduction in the relative weights of all accessory sex organs studied and when injected from 11 to 20 days in epididymis and vas deferens. When cyproterone acetate was injected after 20 days of age, the inhibition of sexual organ weights was reversible and at adulthood organs were normally developed. Cyproterone acetate treatment induced a high percentage of infertile males only when injected from 1 to 10 days. Spermatogenesis, androgen levels in plasma and accessory sex organs, and sexual behaviour were not affected in sterile males. These results suggest that the functional development of accessory sex organs can be permanently affected by short-term neonatal exposure to endogenous androgens.

Protein kinase C pathway potentiates androgen-mediated gene expression of the mouse vas deferens specific aldose reductase-like protein (MVDP).

Transcription of the mouse vas deferens protein (MVDP) gene, a member of the aldo-keto reductase superfamily, is stimulated by androgens via the androgen responsive element (ARE) located in the proximal promoter (-111 to -97). We investigated interaction between androgens and the protein kinase C (PKC) signalling pathway. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT). T47D cells were transiently transfected with 5' flanking MVDP DNA promoter sequences (-1804 to +41; -510 to +41 and -121 to +41) fused to the reporter (CAT) gene. Androgen-induced transcriptional activity can be enhanced from 6 (1.8 and 0.5 kb MVDP-CAT constructs) to 18 fold (0.16 kb MVDP-CAT construct), in a time and dose-dependent manner, by the PKC activator 12-o-tetradecanoylphorbol-13 acetate (TPA). A mutation in the proximal ARE abolished both androgen and TPA-dependent gene enhancement. TPA influenced minimally MMTV promoter in T47D cells and MVDP promoter in CV1 cells suggesting that the effects of the PKC activator are probably promoter and cell-specific. In contrast, activation of protein kinase A (PKA) via addition of dibutyryl-cAMP (db-cAMP) reduced androgen induction of the MVDP gene.