Hydroxysteroid (17ß)-dehydrogenase 1-deficient female mice present with normal puberty onset but are severely subfertile due to a defect in luteinization and progesterone production.

Hydroxysteroid (17ß)-dehydrogenase type 1 (HSD17B1) catalyzes the conversion of low active 17-ketosteroids, androstenedione (A-dione) and estrone (E1) to highly active 17-hydroxysteroids, testosterone (T) and E2, respectively. In this study, the importance of HSD17B1 in ovarian estrogen production was determined using Hsd17b1 knockout (HSD17B1KO) mice. In these mice, the ovarian HSD17B enzyme activity was markedly reduced, indicating a central role of HSD17B1 in ovarian physiology. The lack of Hsd17b activity resulted in increased ovarian E1:E2 and A-dione:T ratios, but we also observed reduced progesterone concentration in HSD17B1KO ovaries. Accordingly with the altered steroid production, altered expression of Star, Cyp11a1, Lhcgr, Hsd17b7, and especially Cyp17a1 was observed. The ovaries of HSD17B1KO mice presented with all stages of folliculogenesis, while the corpus luteum structure was less defined and number reduced. Surprisingly, bundles of large granular cells of unknown origin appeared in the stroma of the KO ovaries. The HSD17B1KO mice presented with severe subfertility and failed to initiate pseudopregnancy. However, the HSD17B1KO females presented with normal estrous cycle defined by vaginal smears and normal puberty appearance. This study indicates that HSD17B1 is a key enzyme in ovarian steroidogenesis and has a novel function in initiation and stabilization of pregnancy.

Seminal vesicles and urinary bladder as sites of aromatization of androgens in men, evidenced by a CYP19A1-driven luciferase reporter mouse and human tissue specimens.

The human CYP19A1 gene is expressed in various tissues by the use of tissue-specific promoters, whereas the rodent cyp19a1 gene is expressed mainly in the gonads and brain. We generated a transgenic mouse model containing a >100-kb 5' region of human CYP19A1 gene connected to a luciferase reporter gene. The luciferase activity in mouse tissues mimicked the CYP19A1 gene expression pattern in humans. Interestingly, the reporter gene activity was 16 and 160 times higher in the urinary bladder and seminal vesicles, respectively, as compared with the activity in the testis. Accordingly, CYP19A1 gene and P450arom protein expression was detected in those human tissues. Moreover, the data revealed that the expression of CYP19A1 gene is driven by promoters PII, I.4, and I.3 in the seminal vesicles, and by promoters PII and I.4 in the urinary bladder. Furthermore, the reporter gene expression in the seminal vesicles was androgen dependent: Castration decreased the expression ∼20 times, and testosterone treatment restored it to the level of an intact mouse. This reporter mouse model facilitates studies of tissue-specific regulation of the human CYP19A1 gene, and our data provide evidence for seminal vesicles as important sites for estrogen production in males.

Estrogen-regulated genes in rat testes and their relationship to recovery of spermatogenesis after irradiation.

Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropin-releasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.

Changes in gene expression in somatic cells of rat testes resulting from hormonal modulation and radiation-induced germ cell depletion.

Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF(1) rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.

Placenta defects and embryonic lethality resulting from disruption of mouse hydroxysteroid (17-beta) dehydrogenase 2 gene.

Hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2) is a member of aldo-keto reductase superfamily, known to catalyze the inactivation of 17beta-hydroxysteroids to less active 17-keto forms and catalyze the conversion of 20alpha-hydroxyprogesterone to progesterone in vitro. To examine the role of HSD17B2 in vivo, we generated mice deficient in Hsd17b2 [HSD17B2 knockout (KO)] by a targeted gene disruption in embryonic stem cells. From the homozygous mice carrying the disrupted Hsd17b2, 70% showed embryonic lethality appearing at the age of embryonic d 11.5 onward. The embryonic lethality was associated with reduced placental size measured at embryonic d 17.5. The HSD17B2KO mice placentas presented with structural abnormalities in all three major layers: the decidua, spongiotrophoblast, and labyrinth. Most notable was the disruption of the spongiotrophoblast and labyrinthine layers, together with liquid-filled cysts in the junctional region and the basal layer. Treatments with an antiestrogen or progesterone did not rescue the embryonic lethality or the placenta defect in the homozygous mice. In hybrid background used, 24% of HSD17B2KO mice survived through the fetal period but were born growth retarded and displayed a phenotype in the brain with enlargement of ventricles, abnormal laminar organization, and increased cellular density in the cortex. Furthermore, the HSD17B2KO mice had unilateral renal degeneration, the affected kidney frequently appearing as a fluid-filled sac. Our results provide evidence for a role for HSD17B2 enzyme in the cellular organization of the mouse placenta.

Deleting the mouse Hsd17b1 gene results in a hypomorphic Naglu allele and a phenotype mimicking a lysosomal storage disease.

HSD17B1 is a steroid metabolising enzyme. We have previously generated knockout mice that had the entire coding region of Hsd17b1 replaced with lacZ-neo cassette (Hsd17b1-LacZ/Neo mice). This resulted in a 90% reduction of HSD17B1 activity, associated with severe subfertility in the knockout females. The present study indicates that Hsd17b1-LacZ/Neo male mice have a metabolic phenotype, including reduced adipose mass, increased lean mass and lipid accumulation in the liver. During the characterisation of this metabolic phenotype, it became evident that the expression of the Naglu gene, located closely upstream of Hsd17b1, was severely reduced in all tissues analysed. Similar results were obtained from Hsd17b1-LacZ mice after removing the neo cassette from the locus or by crossing the Hsd17b1-LacZ/Neo mice with transgenic mice constitutively expressing human HSD17B1. The deficiency of Naglu caused the accumulation of glycosaminoglycans in all studied mouse models lacking the Hsd17b1 gene. The metabolic phenotypes of the Hsd17b1 knockout mouse models were recapitulated in Naglu knockout mice. Based on the data we propose that the Hsd17b1 gene includes a regulatory element controlling Naglu expression and the metabolic phenotype in mice lacking the Hsd17b1 genomic region is caused by the reduced expression of Naglu rather than the lack of Hsd17b1.

Both testosterone and follicle-stimulating hormone independently inhibit spermatogonial differentiation in irradiated rats.

Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.

Spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice with androgen receptor or follicle-stimulating hormone mutations.

The jsd mice experience a single wave of spermatogenesis, followed by an arrest of spermatogenesis because of a block in spermatogonial differentiation. Previous pharmacological and surgical studies have indicated that testosterone (T) and low scrotal temperatures but not FSH block spermatogonial differentiation in jsd mice. We sought to test these observations by genetic approaches by producing male jsd mutant mice with either defective androgen receptor (AR, Tfm mutation) or a deficiency of FSH (fshb(-/-)). In adult jsd-Tfm double-mutant mice, the tubule differentiation index was 95% compared with 14% in jsd littermates, suggesting that general ablation of AR function restored spermatogonial differentiation in jsd mice. The results indicated that this enhancement of differentiation was primarily a result of elevation of temperature caused by the cryptorchid position of the testis in jsd-Tfm double-mutant mice, which resulted from the lack of AR in the gubernaculum. The low levels of T were not a factor in the release of the spermatogonial differentiation block in the jsd-Tfm mice, but we were unable to determine whether inactivation of AR in the adult jsd testis had a direct effect on the restoration of spermatogonial differentiation because the elevated temperature bypassed the T-induced block in spermatogonial differentiation. Although spermatogonia were indeed present in adult jsd-fshb double-mutant mice and were capable of differentiation after androgen deprivation, these mice had a tubule differentiation index of 0%, ruling out the possibility that endogenous FSH inhibited spermatogonial differentiation in jsd mice. The results are consistent in support of the hypothesis that inhibition of spermatogonial differentiation in jsd mice is a result of T acting through the AR only at scrotal temperatures.

Endogenous estrogens inhibit mouse fetal Leydig cell development via estrogen receptor alpha.

It is now accepted that estrogens play a role in male fertility and that exposure to exogenous estrogens during fetal/neonatal life can lead to reproductive disorders in the male. However, the estrogen receptor (ER)-mediated processes involved in the regulation of male reproduction during fetal and neonatal development are still largely unclear. We previously reported that ER beta deficiency affects gametogenesis in mice but changes neither the number nor the differentiated functions of fetal Leydig cells. We show here that ER alpha-deficient mice (ER alpha-/-) display higher levels of testicular testosterone secretion than wild-type mice from fetal d 13.5 onwards. This results from higher levels of steroidogenic activity per fetal Leydig cell, as indicated by the hypertrophy of these cells and the higher levels of mRNA for StAR, P450c17 and P450scc in the testis, for a similar number of Leydig cells. Because LH is not produced on fetal d 13.5 and because no change in plasma LH concentration was observed in 2-d-old ER alpha-deficient mice, LH is probably not involved in the effects of estrogens on testicular steroidogenesis in fetal and early neonatal Leydig cells. Furthermore, inactivation of ER beta did not change the effect of ER alpha inactivation on steroidogenesis. Lastly, in an organ culture system, 1 mum diethylstilbestrol decreased the testosterone secretion of wild-type fetal and neonatal testes but not of ER alpha-/- testes. Thus, this study shows that endogenous estrogens physiologically inhibit steroidogenesis via ER alpha by acting directly on the testis early in fetal and neonatal development.

Pituitary hormones are not required for sexual differentiation of male mice: phenotype of the T/ebp/Nkx2.1 null mutant mice.

We have studied male sexual differentiation of null mutant mice (-/-) for the thyroid-specific enhancer-binding protein (T/ebp or Nkx2.1) gene, a homeodomain transcription factor that plays a role in organogenesis of the thyroid, lung, ventral forebrain, and pituitary gland. Because the T/ebp/Nkx2.1 (-/-) mice do not develop the pituitary gland, their sexual differentiation, if any, must occur in the absence of action of gonadotropins and other pituitary hormones. The (-/-) mice survive only until birth (embryonic d 19-19.5 of pregnancy), and when their external and internal genitals were inspected at embryonic d 18.5, they were indistinguishable from the (+/-) and (+/+) control mice. The testis weights of (-/-) mice were 20% lower than in (+/+) and (+/-) mice. The testosterone content of the (-/-) testes (13.5 +/- 2.4 pg/gonad, mean +/- SEM, n = 11) was dramatically reduced, compared with (+/-) (165 +/- 22.5 pg, n = 14) and (+/+) (234 +/- 37.3 pg, n = 10) littermates. Light microscopy revealed no difference in seminiferous tubules, interstitial tissue, or relative proportions of the two-cell compartments between the (-/-) and (+/+) testes. However, electron microscopy confirmed that Leydig cells in the (-/-) testes were much smaller, with smaller mitochondria and proportion of smooth endoplasmic reticulum than found in the controls, which was in support of the low androgen content of the knockout testes. In conclusion, this study on T/ebp/Nkx2.1 knockout mice, devoid of the pituitary gland, demonstrates that pituitary hormone secretion is not needed for stimulation of sufficient fetal testicular androgen synthesis to induce male sexual differentiation. The endogenous testosterone level in the null mutant testes is 5-10% of the control level, which suggests that there is a considerable safety margin in the amount of testosterone that is needed for the male fetal masculinization.

Failure of normal Leydig cell development in follicle-stimulating hormone (FSH) receptor-deficient mice, but not FSHbeta-deficient mice: role for constitutive FSH receptor activity.

Previous studies have suggested that FSH may be involved in regulation of Leydig cell function. We have examined this directly using two mouse models with null mutations in either the FSH beta-subunit (FSHbetaKO mice) or the FSH receptor (FSHRKO mice). Circulating LH levels were normal in adult FSHbetaKO mice, but were significantly increased in FSHRKO mice. Intratesticular testosterone levels increased normally in FSHbetaKO mice from birth to adulthood, whereas testosterone levels in FSHRKO mice failed to increase normally after puberty and were significantly reduced in adult animals. This was associated with reduced levels of mRNA encoding cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase type VI, and steroidogenic acute regulatory protein in FSHRKO mice. Leydig cell number was normal in FSHbetaKO mice during development, but in FSHRKO mice Leydig cell number increased slowly after puberty and was significantly reduced in the adult animal. Transfection studies showed that the FSHR exhibits constitutive activity in the absence of agonist stimulation. The results indicate, therefore, that Sertoli cells regulate the development of Leydig cell number and that constitutive activity within the FSHR is sufficient to stimulate this process. The presence of the hormone itself is not required when circulating LH levels are adequate.

A Novel mutation in the FSH receptor inhibiting signal transduction and causing primary ovarian failure.

Inactivating mutations of the FSH receptor (FSHR) are known to cause ovarian failure with amenorrhea and infertility in women. The first mutation identified in the FSHR gene was a missense mutation (566C-->T, predicting Ala189Val transition) found in several Finnish patients with primary amenorrhea due to ovarian failure. Only five additional, partially or totally inactivating, mutations of the FSHR have been reported. Here, we report a novel FSHR mutation, 1255G-->A, in a Finnish female with primary amenorrhea. The patient was a compound heterozygote for two mutations in the FSHR gene: 566C-->T, the Finnish founder mutation, and 1255G-->A, a previously unidentified mutation. The new mutation is located in exon 10 in the second transmembrane stretch of the FSHR, and it predicts an Ala419Thr change in the protein structure. In functional testing, the mutation was shown to have minimal effect on ligand binding capacity and affinity, but it almost totally abolished the cAMP second messenger response. Neither of the two FSHR mutations (566C-->T or1255G-->A) was identified in 40 other Finnish patients with premature ovarian failure. Based on this and previous studies, FSHR mutations remain a rare cause of ovarian failure.

Male reproductive effects of octylphenol and estradiol in Fischer and Wistar rats.

4-tert-Octylphenol is a non-ionic surfactant used as a detergent, emulsifier and wetting agent. It is generally accepted that it acts as a weak estrogenic substance when evaluated in in vitro and in vivo short-term screening assays. The sensitivity of animal species (mouse versus rat), strain (inbred versus outbred) has been a matter of concern when selecting assay type for testing of estrogenicity of chemicals. The present study was designed to investigate whether the choice of different animal strain, could affect the outcomes of studies. Fischer and Wistar adult male rats were exposed to vehicle or 400 mg/kg bw of 4-tert-octylphenol administrated orally by gavage. Estradiol benzoate, at a dose of 40 microg/kg bw, was used as positive control agent. Treatment with estradiol benzoate decreased serum levels of testosterone, LH, FSH, inhibin and increased prolactin. Additionally, estradiol benzoate decreased the weight of all investigated reproductive organs, decreased sperm production and increased seminiferous tubular degeneration in both strains. More progressive effects on testis weight and histopathology were observed in the Fischer rats. Oral administration of octylphenol at 400 mg/kg bw to both rat strains increased prolactin levels but had no effect on LH, FSH, testosterone or inhibin. In the octylphenol-treated Fischer rats the weights of the seminal vesicles and the levator ani/bulbocavernosus muscle were significantly decreased, whereas only the levator ani/bulbocavernosus muscle was affected in Wistar rats. The weights of all other reproductive organs and sperm count were unaffected. It is concluded that there might be an organ specific difference in sensitivity between the two strains with the Fischer rat being the most sensitive rat model as demonstrated mainly by the more progressive effects on testis weight and histopathology in estradiol benzoate-treated Fischer rats but also by the decrease in seminal vesicle weight in octylphenol-treated rats.

FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents: a report of the Federation of European Laboratory Animal Science Associations Working Group.

The use of genetically-modified (GM) animals as research models continues to grow. The completion of the mouse genome sequence, together with the high-throughput international effort to introduce mutations across the mouse genome in the embryonic stem (ES) cells (www.knockoutmouse.org) facilitates an efficient way to obtain mutated mouse strains as research models. The increasing number of available mutated mouses trains and their combinations, together with the increasing complexity in the targeting approaches used,reinforces the need for guidelines that will provide information about the mouse strains and the robust and reliable methods used for their genotyping. This information, however, should be obtained with a method causing minimal discomfort to the experimental animals. We have, therefore, compiled the present document which summarizes the currently available methods for obtaining genotype information. It provides updated guidelines concerning animal identification, DNA sampling and genotyping, and the information to be kept and distributed for any mutated rodent strain.

The diversity of sex steroid action: novel functions of hydroxysteroid (17ß) dehydrogenases as revealed by genetically modified mouse models.

Disturbed action of sex steroid hormones, i.e. androgens and estrogens, is involved in the pathogenesis of various severe diseases in humans. Interestingly, recent studies have provided data further supporting the hypothesis that the circulating hormone concentrations do not explain all physiological and pathological processes observed in hormone-dependent tissues, while the intratissue sex steroid concentrations are determined by the expression of steroid metabolising enzymes in the neighbouring cells (paracrine action) and/or by target cells themselves (intracrine action). This local sex steroid production is also a valuable treatment option for developing novel therapies against hormonal diseases. Hydroxysteroid (17ß) dehydrogenases (HSD17Bs) compose a family of 14 enzymes that catalyse the conversion between the low-active 17-keto steroids and the highly active 17ß-hydroxy steroids. The enzymes frequently expressed in sex steroid target tissues are, thus, potential drug targets in order to lower the local sex steroid concentrations. The present review summarises the recent data obtained for the role of HSD17B1, HSD17B2, HSD17B7 and HSD17B12 enzymes in various metabolic pathways and their physiological and pathophysiological roles as revealed by the recently generated genetically modified mouse models. Our data, together with that provided by others, show that, in addition to having a role in sex steroid metabolism, several of these HSD17B enzymes possess key roles in other metabolic processes: for example, HD17B7 is essential for cholesterol biosynthesis and HSD17B12 is involved in elongation of fatty acids. Additional studies in vitro and in vivo are to be carried out in order to fully define the metabolic role of the HSD17B enzymes and to evaluate their value as drug targets.

Mouse testis development and function are differently regulated by follicle-stimulating hormone receptors signaling during fetal and prepubertal life.

It is currently admitted that Follicle-Stimulating Hormone (FSH) is physiologically involved in the development and function of fetal/neonatal Sertoli cells in the rat but not the mouse. However, FSH is produced by both species from late fetal life onwards. We thus reinvestigated the role of FSH in mouse testis development at day 0 (birth) 6, 8 and 10 post-partum (dpp) by using mice that lack functional FSH receptors (FSH-R(-/-)). At birth, the number and proliferative index of Sertoli cells were significantly lower in FSH-R(-/-) mice than in wild type neonates. Claudin 11 mRNA expression also was significantly reduced in FSH-R(-/-) testes at 0 and 8 dpp, whereas the mRNA levels of other Sertoli cell markers (Transferrin and Desert hedgehog) were comparable in FSH-R(-/-) and wild type testes. Conversely, AMH mRNA and protein levels were higher at birth, comparable at 6 dpp and then significantly lower in FSH-R(-/-) testes at 8-10 dpp in FSH-R(-/-) mice than in controls. Although the plasma concentration of LH and the number of Leydig cells were similar in FSH-R(-/-) and control (wild type), testosterone concentration and P450c17 mRNA expression were significantly increased in FSH-R(-/-) testes at birth. Conversely, at 10 dpp when adult Leydig cells appear, expression of the steroidogenic genes P450scc, P450c17 and StAR was lower in FSH-R(-/-) testes than in controls. In conclusion, our results show that 1) like in the rat, signaling via FSH-R controls Sertoli cell development and function during late fetal life in the mouse as well; 2) paracrine factors produced by Sertoli cells are involved in the FSH-R-dependent regulation of the functions of fetal Leydig cells in late fetal life; and 3) the role of FSH-R signaling changes during the prepubertal period.

Hydroxysteroid (17{beta}) dehydrogenase 12 is essential for mouse organogenesis and embryonic survival.

Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) have a significant role in steroid metabolism by catalyzing the conversion between 17-keto and 17beta-hydroxysteroids. However, several studies in vitro have shown that some of these enzymes may also be involved in other metabolic pathways. Among these enzymes, HSD17B12 has been shown to be involved in both the biosynthesis of estradiol and the elongation of the essential very long fatty acids in vitro and in vivo. To investigate the function of mammalian HSD17B12 in vivo, we generated mice with a null mutation of the Hsd17b12 gene (HSD17B12KO mice) by using a gene-trap vector, resulting in the expression of the lacZ gene of the trapped allele. The beta-galactosidase staining of the heterozygous HSD17B12KO mice revealed that Hsd17b12 is expressed widely in the embryonic day (E) 7.5-E9.5 embryos, with the highest expression in the neural tissue. The HSD17B12KO mice die at E9.5 at latest and present severe developmental defects. Analysis of the knockout embryos revealed that the embryos initiate gastrulation, but organogenesis is severely disrupted. As a result, the E8.5-E9.5 embryos were void of all normal morphological structures. In addition, the inner cell mass of knockout blastocysts showed decreased proliferation capacity in vitro, and the amount of arachidonic acid was significantly decreased in heterozygous HSD17B12 ES cells. This, together with the expression pattern, suggests that in mouse, the HSD17B12 is involved in the synthesis of arachidonic acid and is essential for normal neuronal development during embryogenesis.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Hydroxysteroid (17beta) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5-9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.

Sex steroid-dependent and -independent action of hydroxysteroid (17beta) Dehydrogenase 2: evidence from transgenic female mice.

We have recently generated transgenic (TG) mice overexpressing human hydroxysteroid (17beta) dehydrogenase 2 enzyme (HSD17B2TG mice) under the ubiquitous chicken beta-actin promoter. As shown in the present study, the HSD17B2TG female mice presented with slower gain of body weight as compared with the wild-type (WT) littermates and suffered from ovarian dysfunction and mammary gland hyperplasia associated with increased expression of multiple pregnancy-associated genes. The macroscopic phenotype observed in the mammary gland was likely to be dependent on the increased progesterone and prolactin secretion, and a normal histological appearance was observed in HSD17B2TG mammary gland transplanted into a WT host. However, a significant suppression of several known estrogen target genes in the HSD17B2TG mammary transplants in WT females was observed, suggesting that HSD17B2 modulates estrogen action in vivo. Interestingly, the growth retardation of HSD17B2TG females was not efficiently rescued in the bi-TG mice expressing both HSD17B2 and HSD17B1 enzymes, and the bi-TG mice presented with certain masculinized phenotypes, including lack of nipples and closed vagina, recently reported for HSD17B1TG females. The present data suggest that HSD17B2 expression affects both sex steroid-independent and steroid-dependent pathways.

Impact of the chemotherapy cocktail used to treat testicular cancer on the gene expression profile of germ cells from male Brown-Norway rats.

Advances in treatment for testicular cancer that include the coadministration of bleomycin, etoposide, and cisplatin (BEP) have brought the cure rate to higher than 90%%. The goal of this study was to elucidate the impact of BEP treatment on gene expression in male germ cells. Brown-Norway rats were treated for 9 wk with vehicle (0x) or BEP at doses equivalent to 0.3x and 0.6x the human dose. At the end of treatment, spermatogenesis was affected, showing altered histology and a decreased sperm count; spermatozoa had a higher number of DNA breaks. After 9 wk of treatment, round spermatids were isolated, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Of the 31 099 probe sets present on the array, 59%% were expressed in control round spermatids. BEP treatment significantly altered the expression of 221 probe sets, with at least a 1.5-fold change compared with controls; 80% were upregulated. We observed a dose-dependent increase in the expression of oxidative stress response genes and no change in the expression of genes involved in DNA repair. BEP upregulated genes were implicated in pathways related to Jun and Junb protooncogenes. Increased mRNA levels of Jun and Junb were confirmed by quantitative RT-PCR; furthermore, JUN protein was increased in elongating spermatids. Thus, BEP exposure triggers an oxidative stress response in round spermatids and induces many pathways that may lead to the survival of damaged cells and production of abnormal sperm.