Intersex goats show different gene expression levels in the hypothalamus and pituitary compared with non-intersex goats based on RNA-Seq.

The conditions for sex reversal in vertebrate species have been extensively studied, and the results highlighted numerous key factors involved in sex differentiation. However, the transcriptomes in hypothalamic and pituitary tissues from intersex goats have rarely been studied. The aim of this study was to screen candidate genes and signalling pathways related to sex reversal in Huai goats by analyzing gene expression in hypothalamic and pituitary tissues via transcriptome sequencing and bioinformatics analyses. In total, 612 and 139 differentially expressed genes (DEGs) were identified between the intersex and non-intersex groups in the hypothalamus and pituitary, respectively. The DEGs in the hypothalamus and pituitary were significantly enriched in 41 and 16 signalling pathways, respectively, including the calcium signalling pathway, neuroactive ligand-receptor interaction signalling pathway, and oestrogen signalling pathway, which might be related to intersex sex development disorders. A candidate gene from the tachykinin family (TACR1) was significantly enriched in the calcium signalling pathway. Thirty-one DEGs were shared between these two comparisons and were enriched in several acetyl-CoA-related processes and the oestrogen signalling pathway. The results of the real-time PCR analysis show that the transcriptome sequencing results were reliable. The transcriptome data indicate that the regulation of various physiological systems is involved in intersex goat development. Therefore, these results provide helpful data enhancing our understanding of the molecular mechanisms underlying intersex syndrome in goats.

Comprehensive transcriptome analysis of hypothalamus reveals genes associated with disorders of sex development in pigs.

XX sex reversal, also called XX disorders of sex development (XX-DSD), is a condition affecting the development of the gonads or genitalia, and is relatively common in pigs. However, its genetic etiology and transcriptional regulation mechanism in the hypothalamic-pituitary-gonadal axis (HPGA) remain mostly unknown. XX-DSD (SRY-negative) pigs and normal sows were selected by external genitalia observation. The hypothalamus, which is the integrated center of the HPGA was sampled for whole-transcriptome RNA-seq. The role of DEmiRNA was validated by its overexpression and knockdown in vitro. A total of 1,258 lncRNAs, 1,086 mRNAs, and 61 microRNAs differentially expressed in XX-DSD pigs compared with normal female pigs. Genes in the hormone biosynthesis and secretion pathway significantly up-regulated, and the up-regulation of GNRH1, KISS1 and AVP may associate with the abnormal secretion of GnRH. We also predicted the lncRNA-miRNA-mRNA co-expression triplets and constructed three competing endogenous RNA (ceRNA) potentially associated with XX-DSD. Functional enrichment studies suggested that TCONS_00340886, TCONS_00000204 and miR-181a related to GnRH secretion. Further, miR-181a inhibitor up-regulated GNRH1, PAK6, and CAMK4 in the GT1-7 cells. Conversely, transfection of miR-181a mimics obtained the opposite trends. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly higher in XX-DSD gondas than those in normal sows. Taken together, we proposed that the balance of endocrine had broken in XX-DSD pigs. The current study is the first to examine the transcriptomic profile in the hypothalamus of XX-DSD pigs. It provides new insight into coding and non-coding RNAs that may be associated with DSD in pigs.

A Rare Case of Infertility: SRY Positive 46, XX Testicular Disorder of Sexual Differentiation.

Aim To highlight the complexity of infertility causes by describing the rare case of a man with a testicular disorder of sexual differentiation. Diagnosis A 33 years old Caucasian male presented with a 3-year-old history of primary infertility. His investigations revealed a low testosterone and a raised LH and FSH levels. A sample sent for sperm analysis revealed azoospermia. Chromosomal analysis and karyotyping revealed a 46 XX SRY positive karyotype. Treatment The patient was initiated on testosterone replacement and on calcium/vitamin D supplements. Conclusion Fertility evaluation requires complex assessments and a broad knowledge of possible causes.

ErbB4 point mutation in CU3 inbred rats affects gonadotropin-releasing-hormone neuronal function via compromised neuregulin-stimulated prostaglandin E2 release from astrocytes.

Gonadotropin releasing hormone (GnRH)-secretion is not only regulated by neuronal factors but also by astroglia cells via growth factors and ErbB receptors of the epidermal growth factor family. Studies in transgenic mice carrying mutations in the ErbB receptor system experience impaired reproductive capacity. In addition, some of these animals show a typical skin phenotype with wavy hair and curly whiskers. The rat strain SPRD-CU3 (CU3), examined in this study, displays a similar skin phenotype and a significant impairment of the timing of puberty onset and reproductive performance, suggesting a disruption in the astrocytic to GnRH neuronal communication. To address this issue, we analyzed astrocytic prostaglandin E2 (PGE2 ) release from primary hypothalamic astrocytic cell cultures after stimulation with transforming growth factor α (TGFα), ligand for ErbB1/ErbB2, or Neuregulin 1 beta 2 (NRG1ß2 ), ligand for ErbB4/ErbB2 signaling pathway. Compared to cultures from wild type animals, astrocytic cultures from CU3 rats were unable to respond to NRG stimulation, suggesting a disruption of the ErbB4/ErbB2 signaling pathway. This is confirmed by mutational analysis of ErbB4 that revealed a single point mutation at 3125 bp resulting in an amino acid change from proline to glutamine located at the carboxy-terminal region. As a consequence, substantial conformational changes occur in the transmembrane and intracellular domain of the protein, affecting the ability to form a receptor dimer with a partner and the ability to function as a transcriptional regulator. Thus, astroglia to GnRH neuronal signaling via ErbB4 is essential of timely onset of puberty and reproductive function.

Pubertal effects of 17α-methyltestosterone on GH-IGF-related genes of the hypothalamic-pituitary-liver-gonadal axis and other biological parameters in male, female and sex-reversed Nile tilapia.

The influence of 17α-methyltestosterone (MT) on growth responses, biological parameters and the expression of genes involved in the GH-IGF pathway of the hypothalamic-pituitary-liver-gonadal axis were investigated in female, male, and sex-reversed Nile tilapia to evaluate the relationship between sex and MT-induced changes in these parameters. Female fish had a lower growth rate than male and sex-reversed fish, and MT increased growth performance and duodenal villi in females. Most but not all biological parameters of sex-reversed fish were similar to those of male fish. Male fish had higher red blood cell counts and hemoglobin levels than female and sex-reversed fish, suggesting that these hematological indices reflect a higher metabolic rate in male fish. Greater blood triglyceride levels indicated the vitellogenin process in female fish. MT increased the alternative complement activity in female fish (P<0.05). Sex and MT had no significant effects on the hypothalamic mRNAs of GHRH and PACAP. Although not statistically significant, females tended to have higher GH mRNA levels than male and sex-reversed fish. Additionally, MT tended to decrease and increase GH mRNA levels in female and male fish, respectively. There were significant differences among sexes in the expression of GHR, and IGF mRNAs at the peripheral level in the liver and gonads. Females had lower hepatic GHRs and higher ovarian GHRs than male and sex-reversed fish. While the mRNA levels of IGF-1 were lower in the ovary, the levels of IGF-2 were higher compared with those in testes. A significant correlation between GHRs and IGFs was demonstrated in the liver and gonad (except for IGF-1). Multiple regression analysis showed a significant relationship between GH mRNA and both GHRs and IGFs in the liver and gonad. MT exerted androgenic and, to some extent, estrogenic effects on several physiological parameters and GH-IGF action.

Size-dependent sex change can be the ESS without any size advantage of reproduction when mortality is size-dependent.

Almost all models of sex change evolution assume that reproductive rate increases with body size. However, size-dependent sex changing plants often show size-independent reproductive success, presumably due to pollen limitation. Can the observed size-dependent sex change pattern be the ESS in this case? To answer this question, we analyze a game model of size-dependent sex expression in plants. We assume: (1) reproductive rate is perfectly independent of size; (2) mortality decreases with size in the same way for both sexes; (3) growth rates decrease at maturity, more for females than males. We show that the ESS is size-dependent sex expression: small individuals are vegetative, intermediate individuals are male, and large individuals are female. These results demonstrate that mortality is important in size-dependent sex allocation even when mortality rate is independent of sex. They also offer an explanation of why we see populations in poor environments to have sex ratios more biased toward the first sex relative to high quality environments.

Diferenciación sexual: el factor de Jost / Sexual differentiation: the Jost factor

El estudio de la diferenciación sexual de los mamíferos es, sinduda, un ejemplo relevante de proceso epigénetico producido por lainteracción entre genoma y hormonas secretadas por los testículos fetales: la testosterona y la hormona anti-Müllerian. La diferenciación sexual se produce a nivel periférico, en las gónadas y, también, a nivel cerebral, hipotálamico, en dos vertientes: la neuroendocrina y la de conducta sexual. Ambas vertientes del dimorfismo sexual cerebral pueden ser estudiadas en rata. La primera, por la ovulación, o no, que se produce en un ovario trasplantado en la cavidad abdominal de ratas hembras o en machos, los cuales son, ambos, castrados, previamente al nacimiento, y, la segunda, por la postura de lordosis, que presentan las ratas hembras, debidamente diferenciadas sexualmente, frente al macho. Se han localizado las diferentes zonas cerebrales en donde existen receptores para estrógenos. En la diferenciación periférica o gonadal el diferenciador es el testículo fetal que secreta dos hormonas. La testosterona que mantiene y diferencia los canales Wolff en vasos deferentes, epidídimo y vesículas seminales y la hormona anti-Müllerian (AMH) que provoca la regresión de los canales de Müller, todo ello, en el embrión genéticamente macho. En el cual se diferencian, previamente, los testículos en la etapa fetal de desarrollo de gónadas. En el embrión hembra, sin testículos, y consecuentemente sin testosterona ni AMH, los canales de Wolff involucionan y los de Müller, de forma espontánea, se diferencian en útero, trompas de Falopio y parte superior de la vagina. El conocimiento y aclaración de dichas cuestiones pudo establecerse por el descubrimiento, en fetos de conejos, hecho por el Profesor Alfred Jost, en París (1947-50), de la hormona anti-Müllerian (AMH). Actualmente, la AMH se presenta con múltiples funciones, aunque la más fundamental sea la de regresión de los canales Müllerianen los fetos genéticamente masculinos. Esta hormona es, además, un marcador de patologías como las neoplasias ováricas o la anormal esteroidogénesis del ovario y su hallazgo aclara todo el heterogéneo grupo de patologías de intersexualidad gonadal. Se ha clonado su gen y se han preparado sus anticuerpos. Por pertenecer a la familia del TGFβ (factor de crecimiento transformanteβ) cuyos miembros están implicados en procesos neoplásicos está siendo, actualmente, muy estudiada. Tanto sus posibles aplicaciones en terapéutica, como sus funciones en adulto, son aún investigaciones abiertas al futuro (AU)

Sexual differentiation: the Jost factor Sexual differentiation in mammals is a good example of the epigenetic process produced by the interaction between the genome and hormones secreted by the fetal testes: testosterone and anti-Müllerian hormone (AMH).Sexual differentiation takes place in the gonads and brain(hypothalamus) in two branches: neuroendocrine and sexual behavior. Both branches of the cerebral sexual dimorphism can be studied in the rat. The former by the ovulation pattern of an ovary transplanted in the abdominal cavity of male or female rats which are castrated at birth. The latter can be examined by the response of lordosis of female rats with plain sexual differentiation in front ofthe male. The different brain regions containing estrogen receptors have been localized. Fetal testes regulate gonadal differentiation through two hormones; testosterone an anti-Müllerian hormone. Testosterone differentiates Wolff channels in deferent vessels, epididimus and seminal vesicles, and AMH induces the regression of Müller channels in the genetically male embryo, in which testes are previously differentiated in the last fetal stage of gonad development. In the female, with no testosterone or AMH, the Wolff channels undergo involution and those of Müller spontaneously differentiate to uterus, Fallopian trumps and upper part of vagina. The credit for the knowledge of these matters should be given to Prof. Alfred Jost (Paris, 1947-50), who discovered AMH in rabbit fetuses. Currently, AMH has been endowed with many biological functions, the most important being the involution of Müllerian channels in genetically male fetuses. AMH is a biomarker of diseases such as ovarian tumors and abnormal steroid synthesis in ovary and its finding helped explain a heterogeneous number of sexual-related pathologies. AMH gene has been cloned and anti-AMH monoclonal antibodies obtained. Since AMH has been associated with the transforming growth factor beta (TGF-β) family, whose members are involved in cancer processes, its biological functions and potential therapeutic applications are currently and will certainly be subject of intense studies (AU)

Beliefs and practices concerning twins, hermaphrodites, and albinos among the Bamana and Maninka of Mali.

The Bamana and Maninka of Mali greatly value twins, and have elaborated a range of cultural beliefs and practices to assure their survival. Rates of twinning among these two ethnic groups average from 15.2/1000 to 17.9/1000 births compared to 10.5/1000 births (without assisted reproduction) in the United States and Great Britain. Twins (flaniw) are regarded as extraordinary beings with unusual powers, and as a gift from the supreme deity. A small altar (sinzin) is maintained in the home of twins, and periodic sacrifices of chicken blood, kola nuts, millet paste and millet beer regularly made to assure their protection. Albinos (yéfeguéw) and true and pseudo-hermaphrodites (tyéténousotéw) are also considered twin beings. However, they are believed to be the result of aberrant parental social behavior. The Bamana and Maninka believe that all four groups (twins, albinos, hermaphrodites, and pseudo-harmaphrodites) are closely linked to Faro, an androgynous supernatural being who provides equilibrium in the world. Faro is the original albino and hermaphrodite who gave birth to the first pair of twins after self-impregnation. Whenever a twin dies, a small wooden statue is sculpted called a flanitokélé (twin that remains). This commemorative figure is kept close to the surviving twin, reflecting a belief in the inseparability of twins. Eventually, the surviving twin takes responsibility for the figure. When a surviving twin marries, another figure is often sculpted in the opposite sex from the deceased twin, and placed with the original sculpture. Such commemorative sculptures are not created upon the death of those who are albinos, hermaphrodites, or pseudo-hermaphrodites. In recent years, transformational belief patterns have evolved as increasing numbers of Bamana and Maninka embrace Islam. Traditional beliefs are often given Islamic myths of origin. However, even in this Islamic context, many practices that assure twin survival are maintained.

Isolated 17,20-lyase (desmolase) deficiency in a 46,XX female presenting with delayed puberty.

OBJECTIVE: To investigate the cause of hypergonadotropic hypogonadism. DESIGN: Case report and literature review. SETTING: University Departments of Pediatric Endocrinology and Obstetrics and Gynecology. PATIENT(S): A 13.5-year-old girl with absent puberty and growth retardation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Detailed biochemical, radiological, and molecular analysis, including pelvic ultrasound, basal steroid hormone analysis in serum and aspirated follicle fluid, serum steroid measurement after ACTH (Synachten) and human chorionic gonadotropin (hCG) stimulation, and molecular analysis of CYP17. RESULT(S): This girl with hypergonadotropic hypogonadism (LH 65 U/L, FSH 50 U/L) had a 46,XX karyotype, small uterus and enlarged cystic ovaries, and markedly delayed bone age (9 years). Basal (serum, follicular) and stimulated (serum) steroid hormone levels were consistent with isolated 17,20-lyase deficiency whereas relatively normal P and 17-hydroxyprogesterone concentrations were detected together with very low androstenedione, T, and E(2) levels. CONCLUSION(S): Isolated 17,20-lyase deficiency should be considered in the differential diagnosis of hypergonadotropic hypogonadism in 46,XX females, and follicular fluid steroid analysis is a useful adjuvant test. Failure to detect mutations in CYP17 raises the possibility of a novel association of these phenotypes.

Context-dependent autonomous self-fertilization yields reproductive assurance and mixed mating.

The evolution of self-fertilization in hermaphrodites is opposed by costs that decrease the value of self progeny relative to that of outcross progeny. However, self-fertilization is common in plants; 20% are highly selfing and 33% are intermediate between selfing and outcrossing. Darwin proposed an adaptive benefit of self-pollination in providing reproductive assurance when outcrossing is impossible. Moreover, if outcross pollen receipt is inconsistent within or between years, these conditions likewise favour self-pollination, and this can result in a mixture of self and outcross seed production (mixed mating). Despite wide acceptance, the reproductive assurance hypothesis has lacked the support of complete empirical evidence to show that variable pollination can create both the ecological and genetic conditions favouring self-pollination. We recently showed in Collinsia verna that during periods of infrequent pollinator visits, autonomous self-pollination boosted seed output per flower, the key ecological condition. Here we show low inbreeding depression and marker-based estimates of selfing, demonstrating that when the pollination environment in wild populations necessitates reproductive assurance, selfing rates increase. We provide a complete demonstration of reproductive assurance under variable pollination environments and mechanistically link reproductive assurance to intermediate selfing rates through mixed mating.

Variation in restorer genes and primary sexual investment in gynodioecious Plantago coronopus: the trade-off between male and female function.

In many gynodioecious species the nuclear inheritance of male fertility is complex and involves multiple (restorer) genes. In addition to restoring plants from the female (male sterile) to the hermaphrodite (male fertile) state, these genes are also thought to play a role in the determination of the quantity of pollen produced by hermaphrodites. The more restorer alleles a hermaphroditic plant possesses, the higher the pollen production. To test this hypothesis I combined the results of crossing studies of the genetics of male sterility with phenotypic data on investment in stamens and ovules among the progeny of plants involved in these studies. The sex ratio (i.e. the frequency of hermaphrodites among the progeny), being a measure of the number of restorer alleles of the maternal plant, was positively related to the investment in pollen (male function), but negatively related to the investment in ovules (female function), in both field and greenhouse experiments. Consequently, a negative correlation between male and female function was observed (trade-off) and it is suggested that antagonistic pleiotropic effects of restorer genes might be the cause. Phenotypic gender, a measure combining investment in both pollen and ovules, was highly repeatable between field and greenhouse, indicating genetic determination of a more male- or female-biased allocation pattern among the studied plants.

A synthetic review of the theory of gynodioecy.

Cytoplasmic male sterility alleles (CMS) and corresponding nuclear restorer alleles (R) determine gender expression in gynodioecious populations. In this article, we combine cytonuclear epistasis theory, multilevel selection theory, and resource allocation theory to partition the many selective forces acting in gynodioecious species. This description of selective forces reveals that the notion that genomic conflict leads to the spread of restorers is erroneous. By elucidating the effect of each selective force alone and in combination with the others, our approach shows why change in a single parameter, such as the degree of restorer dominance or the cost of restoration, can result in a myriad of opposing selective effects, making it difficult to test theoretical predictions in experimental systems. In particular, our approach allows us to identify the harmonic mean resource allocation between pollen and ovules and the attendant Fisherian selection as playing a critical role in the evolution of the restorer allele and gender polymorphism. We use this conceptual framework to propose empirical methods that emphasize the role of Fisherian selection acting in gynodioecious populations. Because our approach increases the number and specificity of theoretical predictions, we argue that it provides a better framework for empirical testing.

Characterization of two novel homozygous missense mutations involving codon 6 and 259 of type II 3beta-hydroxysteroid dehydrogenase (3betaHSD) gene causing, respectively, nonsalt-wasting and salt-wasting 3betaHSD deficiency disorder.

We identified two homozygous missense mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3/betaHSD) gene, the first in codon 6 of exon II [CTT (Leu) to TTT (Phe)] in a male infant with hyperpigmented scrotum and hypospadias, raised as a male and no apparent salt-wasting since neonatal age, and the second in codon 259 of exon IV [ACG (Thr) to ATG (Met)] in a male pseudohermaphrodite with labial scrotal folds, microphallus, chordee, and fourth degree hypospadias, raised as a female and with salt-wasting disorder since neonatal age. In vitro transient expression of mutant type II 3betaHSD complementary DNAs of L6F, T259M, as well as T259R for comparison was examined by a site-directed mutagenesis and transfection of construct into COS-1 and COS-7 cells. Northern blot analysis revealed expression of similar amounts of type II 3betaHSD messenger ribonucleic acid from the COS-1 cells transfected by L6F, T259M, T259R, and wild-type (WT) complementary DNAs. Western immunoblot analysis revealed a similar amount of L6F mutant protein compared to WT enzyme from COS-1 cells, but neither L6F from COS-7 cells nor T259M or T259R mutant protein in COS-1 or COS-7 cells was detectable. Enzyme activity in intact COS-1 cells using 1 micromol/L pregnenolone as substrate in the medium after 6 h revealed relative conversion rates of pregnenolone to progesterone of 46% by WT enzyme, 22% by L6F enzyme, and 8% by T259M enzyme and less than 4% activity by T259R enzyme. Using 1 micromol/L dehydroepiandrosterone as substrate, the relative conversion rate of dehydroepiandrosterone to androstenedione after 6 was 89% by WT enzyme, 35% by L6F enzyme, 5.1% by T259M enzyme and no activity by T259R enzyme. However, the L6F mutant 3betaHSD activity, despite its demonstration in the intact cells, was not detected in homogenates of COS-1 cells or in immunoblots of COS-7 cells, suggestive of the relatively unstable nature of this protein in vitro, possibly attributable to the decreased 3betaHSD activity. In the case of T259M and T259R mutations, consistently undetectable proteins in both COS cells despite detectable messenger ribonucleic acids indicate severely labile proteins resulting in either no or very little enzyme activity, and these data further substantiate the deleterious effect of a structural change in this predicted putative steroid-binding domain of the gene. In conclusion, the findings of the in vitro study of mutant type II 3betaHSD enzyme activities correlated with a less severe clinical phenotype of nonsalt-wasting and a lesser degree of genital ambiguity in the patient with homozygous L6F mutation compared to a more severe clinical phenotype of salt-wasting and severe degree of genital ambiguity in the patient with homozygous T259M mutation in the gene.

Correlation of clinical, endocrine and molecular abnormalities with in vivo responses to high-dose testosterone in patients with partial androgen insensitivity syndrome.

OBJECTIVE: To investigate the responses of two patients previously diagnosed as Reifenstein's syndrome to graded high-dose testosterone in terms of hormone levels, nitrogen balance and sebum secretion and to attempt to correlate these parameters with the properties of their androgen receptors and mutations in the androgen receptor gene. DESIGN: Nitrogen balance was determined by comparing controlled nitrogen intake to the amount excreted. Sebum excretion was measured on the forehead. Patients were studied during control periods (no treatment) and during administration of testosterone propionate. Blood samples were used as a source of genomic DNA and to measure peripheral hormone levels; androgen receptor binding was determined using genital skin fibroblasts. PATIENTS: Two patients of XY karyotype, with ambiguous external genitalia and problems of testicular descent who had required mastectomy as teenagers. Normal male controls of proven fertility. MEASUREMENTS: Nitrogen balance, sebum excretion rate and peripheral hormone levels (testosterone, dihydrotestosterone, LH and FSH) were studied before and after testosterone therapy (1 or 5 mg/kg/day). Genomic DNA was extracted from peripheral blood leucocytes and regions of the androgen receptor gene amplified by polymerase chain reaction using pairs of specific primers. Mobility of amplified DNA from patients was analysed on denaturing gradient acrylamide gels and fragments differing in mobility from those of normal controls were sequenced. Fibroblasts were cultured from scrotal skin biopsies and androgen receptor binding parameters, subcellular localization and up-regulation were determined. RESULTS: Testosterone therapy resulted in raised plasma testosterone, dihydrotestosterone and oestradiol in both patients. In patient 1 (lesser genital abnormality), LH was suppressed by 5 mg/kg/day testosterone to the upper limit of the normal range but FSH remained low normal. Both LH and FSH were suppressed by testosterone treatment in patient 2 (greater genital abnormality). Nitrogen retention was increased in both patients (4.2 and 3.0 g/24 h respectively); sebum excretion rate increased to normal in patient 1 but showed no change in patient 2. Mutations in the androgen receptor gene were identified in both patients. In patient 1 a single nucleotide change from adenosine to guanosine resulted in the substitution of glycine for glutamic acid at position 772 within the hormone binding domain of the receptor. In patient 2 a single nucleotide mutation from guanosine to adenosine resulted in the substitution of lysine for arginine at position 608 (exon 3) situated in the second zinc finger of the DNA binding domain. Both patients had a normal number of androgen binding sites in genital skin fibroblasts but those in patient 1 showed reduced binding affinity and rapid dissociation of receptor/ligand complexes while those in patient 2 showed defective nuclear localization. CONCLUSION: In patients with partial androgen insensitivity syndrome the type of androgen receptor mutation and responses to short-term androgen treatment can be correlated with the individual's potential to virilize. If there is a mutation in the androgen receptor DNA binding domain the patient may show little ability to virilize either spontaneously at puberty or after androgen treatment. Sebum excretion appears to be more discriminating than nitrogen balance or gonadotrophin suppression as an index of tissue response to androgens.

No mutation in cytochrome P450 side chain cleavage in a patient with congenital lipoid adrenal hyperplasia.

Molecular basis of lipoid adrenal hyperplasia (lipoid CAH) in a Japanese patient was investigated. A 46XY Japanese female patient was clinically diagnosed as having lipoid CAH based on her clinical history of adrenal crisis at birth and the low basal concentrations of cortisol, aldosterone, adrenal androgens and testosterone in serum. In vitro studies of testicular mitochondrial enzymes confirmed a specific impairment of cholesterol side chain cleavage (SCC) activity. However, in spite of the virtual reduction of SCC activity, the amounts of immunodetectable P450scc, adrenodoxin reductase, and adrenodoxin in testicular mitochondria were almost same as those of normal testis. Furthermore, the size of each protein was similar to that of normal testis. Enzymatic amplification of the complementary DNA encoding P450scc from the patient's testis RNA and its nucleotide analysis by direct sequencing revealed no mutation. These results indicate that defective P450scc is not the lesion in this patient, confirming a previous report showing no P450scc mutations in patients with lipoid CAH. The exact lesion causing lipoid CAH in this patient is currently unknown.

Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3.

Defects in the conversion of androstenedione to testosterone in the fetal testes by the enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) give rise to genetic males with female external genitalia. We have used expression cloning to isolate cDNAs encoding a microsomal 17 beta-HSD type 3 isozyme that shares 23% sequence identity with other 17 beta-HSD enzymes, uses NADPh as a cofactor, and is expressed predominantly in the testes. The 17 beta HSD3 gene on chromosome 9q22 contains 11 exons. Four substitution and two splice junction mutations were identified in the 17 beta HSD3 genes of five unrelated male pseudohermaphrodites. The substitution mutations severely compromised the activity of the 17 beta-HSD type 3 isozyme.

A single amino acid substitution (gly743 --> val) in the steroid-binding domain of the human androgen receptor leads to Reifenstein syndrome.

The androgen receptor (AR) from a patient with Reifenstein syndrome (incomplete androgen insensitivity syndrome) was characterized. The patient's pubic skin fibroblasts had normal androgen binding. However, when incubated at 41 C, fibroblasts from the patient had a marked decrease in androgen binding as compared with normal fibroblasts. Analysis of coding sequences of the androgen receptor gene revealed a single nucleotide substitution in exon E, resulting in an amino acid change from glycine (GGG) to valine (GTG) at amino acid 743 within the steroid binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into a human AR complementary DNA followed by expression in COS1 cells led to production of a mutant AR with no significant difference in androgen binding when cells were incubated with androgen at room temperature. However, in contrast to wild type AR expressed in COS1 cells, the mutant AR had markedly lower androgen-binding affinity at 41 C. The mutant receptor could still stimulate a reporter gene at 37 C but this transcriptional stimulation was also decreased when compared with wild type AR receptor in a chloramphenicol acetyltransferase assay. These results suggest that partial androgen resistance in this patient with Reifenstein syndrome is due to a single point mutation in the steroid binding domain of the androgen receptor.

Masculine-liky hypothalamic-pituitary axis in the androgen-insensitive genetically male rat pseudohermaphrodite.

The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 14-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C21 and C19 steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3beta-hydroxy-delta5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male.