Differentiation and development of testis in the oviparous lizard, Calotes versicolor (Daud.)

The differentiation and development of the testis in the lizard Calotes versicolor was studied histologically and histoenzymatically from the day of oviposition (stage 27) to 2 months after hatching. The study reveals the appearance of the gonadal component as a genital ridge at stage 27. The first sign of testis differentiation is observed at stage 33, which displays a well-developed medulla consisting of seminiferous cords comprising Pre-Sertoli cells. The sex differentiation of the embryonic gonads occurs at stage 34. At this stage, seminiferous cords of the testis are prominent and extensive with many pre-Sertoli cells and few spermatogonia. The interstitial space consists of immature fibroblast-type Leydig cells. Pre-Sertoli cells of the seminiferous cords differentiate into Sertoli cells with a triangular nucleus becoming apparent around stages 36-37. The fibroblast-like Leydig cells differentiate into round matured Leydig cells at stage 40. Quantitative estimation of germ cells reveals that the number of germ cells increases in individual gonads, and in 5-day-old hatchling's, this number multiplies by manifold. Spermatogonia show reductional division in the testis of 1-day-old hatchlings.Histochemical localization of Delta5-3beta-HSDH and G-6-PDH activity appears in the seminiferous cords (medulla) of the testis after sexual differentiation (stage 36), indicating that the embryonic medulla is the site of steroidogenesis and not the cortex in C. versicolor. This study also suggests that morphological differentiation of the gonad precedes detectable steroidogenesis in this species. In 10-day-old hatchling's, Delta5-3beta-HSDH activity is seen in the interstitial cells of the testis, which, however, is not detected in the seminiferous tubules. The intensity of the enzyme activity remains more or less the same in the testis up to 10 days after hatching and begins to increase thereafter. The increase in steroidogenesis parallels the progressive post-hatching increase of the interstitial/Leydig cells.     

Female gonad of moles, genus Talpa (Insectivora, mammalia): ovary or ovotestis?

The female gonads of moles (genus Talpa) are composed of a cortex, functioning as an ovary, and a medulla, which is structurally similar to that of the testis. In the female reproductive apparatus there are masculine glandular annexes, such as a bilobate prostate, two Cowper glands, and a penis-like clitoris. All these features have recently led to the hypothesis of the presence of hermaphroditism due to sex-reversal in Talpa. The purpose of this study is to understand the functional significance of the structural organization of the female gonads in order to verify this hypothesis. Histological, histochemical and ultrastructural analyses have been carried out on several gonads of both sexes of two species: T. europaea and T. romana, including three fetuses. In both species, the cortical region of the female gonad shows a regular oogenetic activity. While the medulla is composed of interstitial cells that are partly organized in cord-like structures, no spermatogenetic activity has been ever observed inside of them. A histochemical analysis shows that in both sexes the interstitial cells secrete steroids, presumably estrogens as well as androgens. The presence of androgens in the female gonads would therefore explain the persistence of male glandular annexes in the female reproductive apparatus and both the sexual and behavioral monomorphism typical of the genus Talpa. Nonetheless, the female gonad of moles is a real ovary and a well-defined gonochorism exists. Therefore, there is no reason to assert the presence of hermaphroditism due to sex reversal.     

Role of renal NO production in the regulation of medullary blood flow

The unique role of nitric oxide (NO) in the regulation of renal medullary function is supported by the evidence summarized in this review. The impact of reduced production of NO within the renal medulla on the delivery of blood to the medulla and on the long-term regulation of sodium excretion and blood pressure is described. It is evident that medullary NO production serves as an important counterregulatory factor to buffer vasoconstrictor hormone-induced reduction of medullary blood flow and tissue oxygen levels. When NO synthase (NOS) activity is reduced within the renal medulla, either pharmacologically or genetically [Dahl salt-sensitive (S) rats], a super sensitivity to vasoconstrictors develops with ensuing hypertension. Reduced NO production may also result from reduced cellular uptake of l-arginine in the medullary tissue, resulting in hypertension. It is concluded that NO production in the renal medulla plays a very important role in sodium and water homeostasis and the long-term control of arterial pressure.     

Testicular graft on the chick embryo

A testis from an 18-day-old chick embryo was transplanted into the extra-coelomic cavity of 3-4-day-old hosts. The embryos surviving at 17 days were sacrificed and their genital system was examined. Testis grafting produced inhibition of testicular development. Development of the female gonads was also inhibited. A more or less complete modification of sex was associated with this inhibition. The left ovary lost its cortex, but its medulla remained mostly ovarian in structure. The right gonad frequently acquired a typical testicular structure. These results confirm the possibility of obtaining sex reversal in the female chick embryo by testis grafting.     

Effects of steroid sex hormones on chick embryo gonads in organ culture, with special reference to hormonal control of gonadal sex differentiation

At the initial stages of sex differentiation (7.5 and 8.5 days of incubation), chick embryo gonads were treated directly with testosterone or estradiol-17 beta in organ cultures. Chemically-defined media containing cholesterol as a steroid precursor were used. The differentiation of gonads in the 10 to 12-day controls, cultured in media containing no hormones, was close to that of gonads of equivalent age in ovo. Testosterone added to the medium exerted an inhibitory effect on the cortex of the female gonad and a masculinizing one on its medulla. The results of estradiol treatment confirmed the known feminizing effect of that hormone on the male gonad, the meiotic prophase in the genetically male germ cells being initiated in the induced cortex. These data may be interpreted in favour of a bihormonal theory of gonadal sex differentiation in birds, where the predominantly-synthesized male or female hormone in the gonad determines the male or female pattern of development of the corresponding gonad.     

Absence of correlations between glutamine-synthetase activity and dysmyelination-associated modifications of astroglia in the brain of murine mutants

Glutamine Synthetase (GS) activity was investigated in cerebellum (ce), cerebral cortex (cc), olfactory bulb (ob), and medulla oblongata (mo) of murine dysmyelinating mutants for correlations with modifications of astroglia associated with genetic dysmyelination. One of these mutants, jimpy, develops a strong gliosis throughout the CNS. The other three mutants: shiverer, mld, and quaking, exhibit various astrocytic responses to dysmyelination, but reduced gliosis if any. Comparison between CNS areas in control animals showed a higher GS activity in the olfactory bulb than in the cerebral cortex, medulla, and cerebellum. The developmental patterns of GS activity were similar in mutants and in controls in all four areas investigated. Data on Jimpy suggest that GS activity is not associated with reactive astrocytes.     

Developmental endocrinology of the reproductive axis in the chicken embryo

In mammals, the phenotype of the homogametic sex develops in the (relative) absence of steroids and the phenotype of the heterogametic sex is imposed by the early action of steroids. In contrast, the heterogametic sex in avian species is the female and the presence of estrogens and their receptors plays a crucial role in female sexual differentiation. The time- and sex-dependent expression of enzymes involved in steroidogenesis which determine the ratio of androgens/estrogens produced by the gonads has been extensively investigated during the last 5-6 years. These results all show that the lack of estrogen synthesis in the male appears to be due to the extremely low levels of 17beta-hydroxysteroid dehydrogenase and P450aromatase expression. In females, extensive expression of the aromatase gene (around day 5-6 of incubation), leading to estrogen synthesis, and specific expression of the estrogen receptor-mRNA in the left gonad results in the development of a functional left ovary. Other sex differences can be found in the expression of the inhibin subunit genes in gonads of chicken embryos and in circulating concentrations of inhibin, follicle stimulating hormone (FSH) and steroids. Sex reversal attempts have been made by varying incubation temperatures, by using anti-estrogens, androgens, aromatase inhibitors and synthetic steroids. In ovo administration of a sex steroid hormone or an inhibitor of endogenous sex steroid synthesis can cause phenotypical sex reversal. All these experiments show that the development of gonads in birds is very sensitive to changes in the embryonic hormonal environment, sometimes resulting in changes of postnatal reproduction and even growth.     

Regional enzyme development in rat brain. Enzymes of energy metabolism.

The development of several key enzymes of pyruvate and 3-hydroxybutyrate metabolism and of the tricarboxylic acid cycle was studied in six regions (cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex) of the neonatal, suckling and adult rat brain (2 days before birth to 60 days after birth). The enzymes whose developmental patterns were studied were: pyruvate dehydrogenase (EC 1.2.4.1), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and fumarase (EC 4.2.1.2). Citrate synthase, isocitrate dehydrogenase and pyruvate dehydrogenase develop as a cluster in each region, although the pyruvate dehydrogenase appears to lag slightly behind the others. As with the glycolytic-enzyme cluster [Leong & Clark (1984) Biochem. J. 218, 131-138] the timing of the development of the activity of this group of enzymes varies from region to region; 50% of the adult activity developed first in the medulla oblongata, followed by the hypothalamus, striatum and mid-brain, and then in the cortex and cerebellum respectively. The 3-hydroxybutyrate dehydrogenase activity also develops earlier in the medulla oblongata than in the other regions. The results are discussed with respect to the neurophylogenetic development of the brain regions studied and the importance of the development of the enzymes of aerobic glycolysis in relationship to the development of neurological maturation.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

Steroid regulation of monoamine oxidase activity in the adrenal medulla

Administration of different steroid hormones in vivo has distinct and specific effects on the MAO activity of the adrenal medulla. In an effort to reconstitute these effects in defined cells, we have isolated endothelial cells and chromaffin cells from the bovine adrenal medulla and tested each cell type for sensitivity to these steroids. As in the intact animal, we found that endothelial cell MAO activity was stimulated 1.5- 2.5-fold by 10 microM progesterone, hydrocortisone, and dexamethasone, inhibited by ca. 50% by 17-alpha-estradiol, but unaffected by testosterone. The type of MAO in the endothelial cells was found to be exclusively of the A type. The chromaffin cells had MAO B exclusively and were inert to treatment with dexamethasone. The mode of action of the various steroids on MAO A activity in endothelial cells seemed to be that of affecting the number of MAO molecules, as binding of [3H]pargyline, an MAO inhibitor, changed in proportion to changes in enzyme activity. Consistently, the kinetic parameters for MAO A showed changes in Vmax but not Km under all conditions. The specificity of steroid action on MAO A activity was also supported by the fact that steroid-induced changes in total cell division ([14C]thymidine incorporation) and total protein synthesis ([14C]leucine incorporation) were seen after changes in MAO A. We conclude that the differential effects of steroids on MAO activity in the intact adrenal medulla can be reproduced in cultured adrenal medullary endothelial cells but not in chromaffin cells. Therefore we suggest that the action of these steroid hormones on the intact adrenal medulla may be restricted to the endothelial cell component of this tissue.     

中国大鲵生殖腺胚后发育的形态学观察

为探寻中国大鲵(Andrias davidianus)生殖腺胚后发育的特点及规律,采用解剖学与组织学技术对其形态结构变化进行了观察.结果表明,大鲵的原始生殖腺开始出现于出膜28～49 d;出膜133～175 d时一些个体生殖腺内已初步分化出原始卵泡;出膜259 ～343 d时一些个体生殖腺内已初步分化出生精小叶;出膜427 d时,卵巢已明显分化为皮质与髓质,且髓质内出现了卵巢腔,精巢内生精小叶及其内的腔隙、精巢间质等分化已较为明显;出膜511 d时精巢分化为明显的生精小叶和非成熟小叶两个区域.本文认为,大鲵与其他无羊膜类原始生殖腺的分化一般发生在胚后阶段,而且雌性的分化时间早于雄性.     

Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos

Abstract. In the turtle Emys orbicularis , sexual differentiation of gonads is temperature-dependent. Oestrogens have been shown to be involved in this phenomenon and temperature has been expected to act, directly or indirectly, on regulation of synthesis or activity of cytochrome P-450 aromatase (P-450 arom). We have studied the effects of temperature shifts and of exposure at female- or male-producing temperatures for different times on gonadal aromatase activity and gonadal structure. In a first series of experiments, eggs were incubated at 25°C (masculinizing temperature) up to stage 18 and then exposed for 1 to 8 days at 35°C, a highly feminizing temperature. The response was exponential: aromatase activity increased clearly only after 4 day exposure at 35°C, then it was considerably enhanced. After 1 and 2 days at 35°C, the structure of gonads was not modified. With longer exposures at 35°C, gonads were progressively feminized: medullary epithelial cords disappeared, whereas an ovarian cortex was forming. In another type of experiment, eggs incubated at 30°C (feminizing temperature) until stage 19 were transferred at 25°C for 6 days. In embryos of these shifted eggs, gonadal aromatase activity was about ninefold lower than that in control embryos (maintained at 30°C). However, this activity did not fall to the level measured in embryos of the same stage incubated at 25°C from egg-laying and was about twofold higher than that measured at the time of transfer. Gonads exhibited a cortex anlage but the medulla was more voluminous than that of controls and epithelial cords were beginning to form within. Together these results show that changes in gonadal aromatase activity and in gonadal structure are correlated, and that temperature acts on regulation of P-450 arom synthesis. Amplification of this synthesis during the thermosensitive period at higher temperatures could reflect amplification of expression of the P-450 arom gene.     

Interaction between sodium ion and non-electrolytes in the countercurrent systems of the kidney

A change in the electrolyte concentration in a multicomponent system will in general change the activity of non-electrolyte species, a particular example being the salting-out of dissolved gases (although for some solutes a salting-in effect occurs). It follows that countercurrent multiplication of sodium ions in the renal medulla will inevitably be associated with change in activity of non-electrolytes since this process is characterized by two orthogonal [Na ⁺] gradients in the multiplier loop, the one longitudinal, the other transverse. Using a single loop model we show that the two [Na+] gradients act independently to produce opposing effects on activity so that given, say, a salting-out effect, the longitudinal gradient effects an increase in activity and the transverse a decrease, both being dependent on the magnitude of the interaction. In the latter case, since attenuation results from direct involvement of the solute in the countercurrent multiplication process, the diffusion coefficient of necessity also has a positive influence. In the non-steady state we show that a measure of the time taken to reach equilibrium varies with the square of the transit time of tubular fluid. This means that there are likely to be transient activity differences between the two countercurrent systems, active and passive, in the medulla. Urine appears to be in equilibrium with the inner medulla and so will reflect these activity changes in the steady and non-steady state. This analysis is consistent with previous experimental findings on urinary inert gases. These results could suggest an indirect method of monitoring countercurrent multiplication of sodium. There are also implications for clinical studies since it is often assumed that urine reflects blood concentrations of biologically inert solutes.     

Three-Dimensional Imaging of Prox1-EGFP Transgenic Mouse Gonads Reveals Divergent Modes of Lymphangiogenesis in the Testis and Ovary

The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.     

lon-1 regulates Caenorhabditis elegans body size downstream of the dbl-1 TGF beta signaling pathway

Although the primitive vasculature is identical in XX and XY genital ridges until 11.5 days postcoitum (dpc), by 12.5 dpc the XY gonad develops a distinct vasculature. This male-specific vasculature, which includes the development of a large coelomic vessel, develops coincident with expression of Sry and formation of testis cords. We show that similar levels of proliferation and vasculogenesis expand the primary vasculature in XX and XY gonads. However, soon after Sry expression begins, the XY gonad recruits a large number of endothelial cells from the adjacent mesonephros, a mechanism totally absent in XX gonads. These migrating cells do not contribute to venous or lymphatic development. Instead, these cells contribute to the arterial system, as indicated by expression of ephrinB2 and by elements of the Notch signaling pathway. This newly formed arterial system establishes a new pattern of blood flow in the XY gonad, which we speculate may have an important role in export of testosterone to masculinize the XY embryo.     

Ultrastructural Observations on the Origin and Differentiation of Somatic Cells during Gonadal Development in the Frog Rana nigromaculata

The process of gonad development in the frog Rana nigromaculata was observed using the electron microscope. The gonadal medulla was formed by the proliferation and displacement of the epithelial cells within the primordial gonad, and a distinct continuity was observed between the cortical and medullary cells. Sex differentiation of the gonad occurred directly from the sexually indifferent primordial gonads. In the rudimentary testes, the continuity between the cortical and medullary regions increased closer, and the intermingling of cortical and medullary cells was evident. The inner region of the cortex developed into a cord-like structure and subsequently differentiated into rudimentary seminiferous tubules. The medulla differentiated into the testicular rete and efferent duct. In the rudimentary ovaries, the cortex and medulla were separated and the ovarian cavity was formed in the medullary region. In the cortex, the cortical cells surrounding oocytes which had reached the diplotene stage, differentiated into follicular cells. The intrusion of mesenchymal or blastemal cells derived from extragonadal regions into the cortex or medulla was never observed. These findings do not support Witschi's cortico-medullary antagonistic theory of sex differentiation.     

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.     

The Action of Pitressin on Solute Permeability of the Rabbit Nephron in Vivo

The isotopic equilibration of urea, thiourea, and inulin between urine and plasma was determined in rabbits in the presence or absence of antidiuretic hormone (ADH). Animals were anesthetized with ethanol and permitted to reach steady state after completion of surgery. Tracer was then administered by intraarterial infusion in such a manner that a high constant specific activity in plasma was rapidly attained. Urine flow was kept independent of ADH by addition of mannitol. Urea/creatinine clearance ratios and the accumulation of urea in renal medulla and papilla also remained unaffected by ADH. Under these conditions, thiourea and inulin at all times approached equilibrium, at similar rates. In the absence of ADH, urea also equilibrated at a rate similar to that of inulin. The addition of ADH, however, significantly prolonged the delay before urinary urea reached the high constant specific activity of plasma urea. These observations are interpreted in terms of a specific effect of the hormone on the solute permeability of the nephron.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.