Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry

Gonadal differentiation involves a complex interplay of developmental pathways. The sex determining region Y (SRY) gene plays a key role in testis determination, but its interaction with other genes is less well understood. Abnormalities of gonadal differentiation result in a range of clinical problems. 46,XY complete gonadal dysgenesis is defined by an absence of testis determination. Subjects have female external genitalia and come to clinical attention because of delayed puberty. Individuals with 46,XY partial gonadal dysgenesis usually present in the newborn period for the valuation of ambiguous genitalia. Gonadal histology always shows an abnormality of seminiferous tubule formation. A diagnosis of 46,XY true hermaphroditism is made if the gonads contain well-formed testicular and ovarian elements. Despite the pivotal role of the SRY gene in testis development, mutations of SRY are unusual in subjects with a 46,XY karyotype and abnormal gonadal development. 46,XX maleness is defined by testis determination in an individual with a 46,XX karyotype. Most affected individuals have a phenotype similar to that of Klinefelter syndrome. In contrast, subjects with 46,XX true hermaphroditism usually present with ambiguous genitalia. The majority of subjects with 46,XX maleness have Y sequences including SRY in genomic DNA. However, only rare subjects with 46,XX true hermaphroditism have translocated sequences encoding SRY. Mosaicism and chimaerism involving the Y chromosome can also be associated with abnormal gonadal development. However, the vast majority of subjects with 45,X/46,XY mosaicism have normal testes and normal male external genitalia.     

PCBs as environmental estrogens: turtle sex determination as a biomarker of environmental contamination

Polychlorinated biphenyls (PCBs) are widespread, low-level environmental pollutants associated with adverse health effects such as immune suppression and teratogenicity. There is increasing evidence that some PCB compounds are capable of disrupting reproductive and endocrine function in fish, birds, and mammals, including humans, particularly during development. Research on the mechanism through which these compounds act to alter reproductive function indicates estrogenic activity, whereby the compounds may be altering sexual differentiation. Here we demonstrate the estrogenic effect of some PCBs by reversing gonadal sex in a reptile species that exhibits temperature-dependent sex determination.     

Mammalian sex determination: from gonads to brain

In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior.     

Hypergonadotropic secondary amenorrhea: clinical analysis of 126 cases

OBJECTIVE: To study the etiology and early diagnosis of hypergonadotropic amenorrhea and to explore the appropriate treatment for preserving their reproductive function. METHODS: 126 cases of secondary amenorrhea with serum follicular stimulating hormone (FSH) levels > or = 40 IU/I, were analysed. Their clinical manifestations, karyotypes, ovarian morphology and histology, reproductive hormone assays, and responses to estrogen therapy and ovulation induction were studied. RESULTS: 6 cases presented with histories of ovarian surgery, radiotherapy or chemotherapy. Among the other 120 cases, 18 manifested amenorrhea before or at 25 years of age, 102 developed amenorrhea after age 25. In the former group, 16 (88.9%) showed unilateral or bilateral gonadal dysgenesis, and the other 2(11.1%) were defined as resistant ovaries. Abnormalities of sex chromosome karyotype occurred in 44.4% (8/ 18). In the latter group, 68 underwent laparotomy or laparoscopy examination. Morphological and histological examinations of both ovaries showed atrophic ovaries in all cases accompanied by 30.9% (21/ 68) unilateral gonadal dysgenesis; sex chromosomal abnormality was found in only one with no sexual immaturation. The efficacy of estrogen treatment was significantly better among cases with amenorrhea less than 1 year as compared with those longer than 1 year. Clomiphene challenge test given to 8 cases during their irregular menstrual stages produced an elevation of FSH levels to > 20 IU/I. without any response of estradiol secretion. CONCLUSIONS: The earlier estrogen therapy is initiated, the greater possibility of pregnancy will be achieved in cases suffering from hypergonadotropic amenorrhea. The clomiphene challenge test may provide evidence of waning ovarian function for early diagnosis.     

Zfx mutation results in small animal size and reduced germ cell number in male and female mice

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been speculated to function in sex differentiation, spermatogenesis, and Turner syndrome. We derived Zfx mutant mice by targeted mutagenesis. Mutant mice (both males and females) were smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals were fully masculinized, with testes and male genitalia, and were fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals were fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells was reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibited a growth deficit evident at embryonic day 12.5, which persisted throughout postnatal life and was not complemented by the Zfy genes. These phenotypes provide the first direct evidence for a role of Zfx in growth and reproductive development.     

Sex differentiation -- gonadogenesis and novel genes

During embryogenesis, most organ rudiments differentiate into only one type of organ and functional mutations are normally lethal for the embryo. However, the indifferent gonad has two options, to form either a testis or an ovary, and mutations of this tissue usually produce sex reversal or sterility which is not lethal for the individual. Therefore, gonadal development serves as an excellent model system for investigating questions of cell fate and organogenesis. The studies of human patients showing different types of sex reversal, in combination with the use of transgenic mice and/or gene targeting disruption, have led to the isolation of several genes important for sex development. These include SRY/Sry, encoding the testis-determining factor, Ftz-F1 encoding steroidogenic factor 1 (SF-1) and Wilms' tumor gene (WT-1). However, the mammalian sex differentiation pathway requires the function of a number of additional genes which we are now trying to identify with the help of mRNA differential display technique.     

Ovarian Sertoli-Leydig cell tumor: a SRY gene-independent pathway of pseudomale gonadal differentiation

Sertoli-Leydig cell tumors (SLCT) are rare sex-cord stromal tumors of the ovary composed of undifferentiated gonadal stromal cells, Leydig cells (LC), and Sertoli cells (SC), with the latter forming structures resembling fetal testicular tubules. The histogenetic basis of morphological male differentiation patterns in females is controversial. Here, we report a SLCT with intermediate differentiation in a 23-year-old woman investigated by light microscopy, immunohistochemistry for intermediate filaments, and sex steroid hormone receptors (SSHR), as well as by polymerase chain reaction (PCR) for the presence of the sex-determining region Y gene (SRY). Our investigation shows that the SCs of SLCT express progesterone and androgen receptors as well as cytokeratins and vimentin. By PCR, SLCT-derived genomic DNA lacked the SRY gene, indicating that the SLCT results from a SRY gene-independent pathway of pseudomale gonadal differentiation. The expression of progesterone receptors (PRs) in the SCs of the SLCT is in contrast to their absence in testicular SCs, but in line with their presence in ovarian granulosa and surface epithelial cells. Thus, our results provide strong evidence for a close histogenetic relationship between the SLCT and the female gonocyte-supporting cell, the granulosa cell (GC).     

Preoperative and intraoperative radioimmunodetection of cancer pretargeted by biotinylated monoclonal antibodies

OBJECTIVES: Two cases of 46,XX true hermaphroditism were analyzed for two Y-DNA sequences, including the recently cloned gene for male testis determination, the sex-determining region of the Y chromosome (SRY). METHODS: Polymerase chain reaction was performed to amplify the SRY. DNA was prepared from peripheral blood lymphocytes as well as from gonadal tissue preserved in a paraffin block. RESULTS: One hermaphrodite contained the SRY sequences in peripheral blood lymphocytes and the testicular part of ovotestis tissue preserved in a paraffin block, while in the second patient these sequences were not detected. CONCLUSIONS: The SRY positive subject resulted from occult Y mosaicism rather than from X-Y translocation. Testis differentiation in the SRY negative subject may have been caused by mutation of a gene on the X chromosome or, alternatively, on an autosome.     

Sexual and gonadal dysfunction in adrenal disorders

Among various diseases of the adrenals, major disorders that cause sexual and gonadal disturbances are congenital adrenal hyperplasia(CAH) and Cushing's syndrome, and the others include virilizing or feminizing adrenocortical tumors. CAH was reviewed based on the recent advances in molecular genetics. The most striking discovery was steroidogenic acute regulatory protein, mutations of which produce lipoid adrenal hyperplasia that was previously attributed to P-450scc deficiency. Reversible amenorrhea or impotence is found in patients with Cushing's syndrome. Low plasma estrogen and testosterone levels are associated with female and male patients, respectively. Elevated adrenal androgen accounts for mild virilization in female patients with ACTH-dependent subtypes. The sites of action at which hypercortisolemia suppresses the hypothalamic-pituitary-gonadal axis were discussed.     

Cellular and molecular changes during sex differentiation of embryonic mammalian gonads

Structural and molecular changes during sex differentiation and development of mammalian gonads from early embryonic phase until sexual maturity have been studied by morphologic and immunocytochemical methods in vivo and in experimental culture. The strategy has been to identify cellular macromolecules whose genes are differently expressed in the two sexes and to formulate a hypothetical regulatory chain of sex determination. This approach should provide new possibilities for finding the missing links between the final structural genes and the early regulatory genes, which are differentially expressed before and during gonadal differentiation. On the basis of accumulated structural and molecular evidence, the early epithelial differentiation from the precursor cells via cell aggregates to testicular cords or ovarian follicles is not sexually regulated. The biological consequences of sex determination in the differentiation of the genital organs include changes in the pattern formation of the gonadal epithelia and concomitant alterations in the synthesis and organization of the structural macromolecules.     

Improved preparation of Xenopus oocytes for patch-clamp recording

The testis-determining gene SRY (sex determining region, Y) is located on the short arm of the Y chromosome and consists of a single exon, the central third of which is predicted to encode a conserved motif with DNA binding/bending properties. We describe the screening of 26 patients who presented with 46,XY partial or complete gonadal dysgenesis for mutations in both the SRY open reading frame (ORF) and in 3.8 kb of Y-specific flanking sequences. DNA samples were screened by using the fluorescence-assisted mismatch analysis (FAMA) method. In two patients, de novo mutations causing complete gonadal dysgenesis were detected in the SRY ORF. One was a nonsense mutation 5' to the HMG box, whereas the other was a missense substitution located at the C terminus of the conserved motif and identical to one previously detected in an unrelated patient. In addition, two Y-specific polymorphisms were found 5' to the SRY gene, and a sequence variant was identified 3' to the SRY polyadenylation site. No duplications of the DSS region in 20 of these patients were detected.     

Rapid sequence evolution of the mammalian sex-determining gene SRY

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.     

Psychoneuroendocrinology of depression. Hypothalamic-pituitary-gonadal axis

Women are more susceptible than men to depression, particularly during periods of rapid fluctuation of gonadal hormones, such as premenstrually, postpartum, and during the climacteric. This review summarizes the evidence for the association of depression with abnormalities in reproductive hormones. Although there are similarities in stress hormones changes between depressed women and women with stress-related amenorrhea, no abnormalities in LH activity have been documented in depression. Similarly no abnormalities in LH, estradiol, or progesterone have been documented in premenstrual syndrome (PMS), although complete elimination of monthly cycling with leuprolide improves mood. Some studies have suggested beneficial effects of estrogen on mood in postmenopausal women but as yet there have been no adequately controlled studies of estrogen treatment of either premenopausal or postmenopausal women.     

Chronic renal failure in infants: effect of strict conservative treatment on growth

Two female sibs of first cousin Iranian parents were found to have the syndrome of spastic paraplegia, optic atrophy with poor vision, microcephaly, and normal cognitive development. Karyotype analysis showed a normal female constitution in one and a male constitution (46,XY) in the other. The XY female showed normal female external genitalia, normal uterus and tubes, and streak gonads. SRY gene sequencing was normal. We conclude that the present family probably represents a new autosomal recessive trait of pleiotropic effects including XY sex reversal and adds further evidence for the heterogeneity of spastic paraplegia syndromes as well as sex reversal syndromes.     

Phenotypic manifestations during the development of the dominant and default gonads in mammals and birds

The dominant embryonic gonad--testis in mammals and ovary in birds--secretes one or more morphogenetic substances that exert a major effect on the phenotype of the embryo. When deprived of their gonads, mammalian embryos develop into females, and avian embryos assume predominantly male characteristics, although retaining both oviducts. In order to fulfill their task of masculinizing the reproductive tract, mammalian testes grow and differentiate faster than ovaries. In birds the pattern is less straightforward. In 5-day-old embryos of White Leghorn chickens, sexual differentiation manifests itself in two different ways: (1) the gonads of ZZ embryos are larger, and on day 6 contain more protein and DNA than those of ZW embryos; (2) in both sexes, left gonads are larger than right gonads and contain a thick "germinal epithelium" capable of giving rise to an ovarian cortex under the influence of oestrogen. The pattern changes in embryos aged between 7 and 8 days, when the left gonad of ZW embryos outgrows all others, developing into an ovary, and when the bilateral asymmetry between left and right gonads increases in female embryos. A remnant of gonadal bilateral asymmetry is seen in the distribution of gonads in cases of true hermaphroditism in humans and other mammals. Whereas the initial fast growth of the mammalian testis is assumed to be due to one or more Y-chromosomal genes, that of the early avian testis is mostly simply explained as the effect in the disomic state of one or more genes on the Z chromosome. However, the later growth of the avian ovary is more likely to be due to oestrogen than to a direct gene effect. It is postulated that oestrogen has lost its power to determine ovarian development in mammals, in which both sexes are exposed to the oestrogen-rich environment of the uterus. Hence, the task of sex determination devolves on the fetal testis, whose early development and hormonal function are required to induce the male phenotype, the female phenotype arising in default mode.     

SRY and karyotypic status of one abnormal and two intersexual marsupials

An intersexual agile wallaby (Macropus agilis) with a penis, a pouch and four teats had a sex-chromosome constitution of XXY in lymphocytes and cultured fibroblasts; the sex-determining region Y (SRY) gene was present, consistent with the presence of a testis. An intersexual eastern grey kangaroo (Macropus giganteus) with a small empty scrotum and no penis, and an abnormal red kangaroo (Macropus rufus) with no penis, pouch or teats, both had XX sex-chromosome complements; the SRY gene was not present, consistent with testis absence. The agile wallaby and grey kangaroo described here provide further evidence that scrotal development in marsupials is independent of the Y chromosome. The cause of the abnormalities in the XX individuals cannot be determined until candidate genes are identified. These animals provide a basis for further genetic studies into marsupial intersexuality and sex differentiation.     

Three novel SRY mutations in XY gonadal dysgenesis and the enigma of XY gonadal dysgenesis cases without SRY mutations

Mutations in the Y-located testis-determining gene SRY are one cause for XY sex reversal. We have previously identified four SRY mutations in a total of 45 sex-reversed females with XY gonadal dysgenesis (XY GD). In a new sample of 16 XY GD cases, three previously undescribed SRY mutations were identified. Two are point mutations that lead to amino acid substitutions in the HMG domain of SRY, M64R, and F67V. The third SRY mutation is a single base insertion 5' to the HMG box within codon 43, converting this lysine codon to a stop codon (K43X). A total of 33 SRY mutations have so far been described that account for only 10-15% of XY GD females. A further 10-15% of these cases result from deletion of SRY due to aberrant X/Y interchange. The etiology of the remaining 70-80% of XY GD cases is still enigmatic. Possible explanations for these XY sex-reversal cases are discussed.     

The basal concentration and characteristics of the diurnal secretory rate of prolactin in women with primary hypothyroidism]

Basal concentration was measured of prolactin (PRL) as were its diurnal fluctuations in blood depending on the clinical association of primary hypothyrosis (PH) with lactorrhea-amenorrhea, lactorrhea only or lactorrhea associated with disturbances in menstrual and reproductive functions that differ from those in amenorrhea, and without such disturbances. Patients with lactorrhea-amenorrhea syndrome present with continual hyperprolactinemia and no physiological diurnal rythm of PRL secretion. In patients presenting without amenorrhea, and basal hyperprolactinemia, lactorrhea and disordered menses result from transitory "night-time" hyperprolactinemia. PH patients who do not present with lactorrhea and disordered menstrual and reproductive function show physiological diurnal rythm of PRL secretion, with its parameters corresponding to those in healthy women. The development of hyperprolactinemia in PH is not age-related; neither is it associated with severity of hypothyrosis.