Studies of gonadal sex differentiation

Gonadal differentiation has a determinative influence on sex development in human embryos. Disorders of sexual development (DSD) have been associated with persistent embryonal differentiation stages. Between 1998 and 2015, 139 female patients with various (DSD) underwent operations at the Scientific Center of Obstetrics, Gynaecology and Perynatology in Moscow, Russia. Clinical investigations included karyotyping, ultrasound imaging, hormonal measurement and investigations of gonadal morphology. The male characteristics in the embryo are imposed by testicular hormones. When these are absent or inactive, the fetus may be arrested at between developmental stages, or stay on indifferent stage and become phenotypically female. A systematic analysis of gonadal morphology in DSD patients and a literature review revealed some controversies and led us to formulate a new hypothesis about sex differentiation. Proliferation of the mesonephric system (tubules and corpuscles) in the gonads stimulates the masculinization of gonads to testis. Sustentacular Sertoli cells of the testes are derived from mesonephric excretory tubules, while interstitial Leydig cells are derived from the original mesenchyme of the mesonephros. According of the new hypothesis, the original mesonephric cells (tubules and corpuscles) potentially persist in the ovarian parenchyma. In female gonads, some mesonephric excretory tubules regress and lose the tubular structure, but form ovarian theca interna and externa, becoming analogous to the sustentacular Sertoli cells in the testis. The ovarian interstitial Leydig cells are derived from intertubal mesenchyme of the mesonephros, similar to what occurs in male gonads (testis). Surprisingly, the leading determinative factor in sexual differentiation of the gonads is the mesonephros, represented by the embryonic urinary system.     

Extracellular matrix permits the expression of von willebrand’s factor,uptake of Di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels

Summary Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI₂) and von Willebrand’s factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-α mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI₂ by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1μM) substantially increased PGI₂ secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ. Supported by grant 1 RO1 NS2822501 from National Institutes of Health, Bethesda, MD.     

Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different “nuclear landscape” in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell‐type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML‐defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display ～1–3 large PML structures of two morphological types: long linear “rods” or elaborate “rosettes”, which lack substantial SUMO‐1, Daxx, and Sp100. These occur primarily between Day 0–2 of differentiation and become rare thereafter. PML rods may be “taut” between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a “gap” in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML‐defined structures. J. Cell. Biochem. 107: 609–621, 2009. © 2009 Wiley‐Liss, Inc.     

Wound phloem in transition to bundle phloem in primary roots ofPisum sativum L.

Summary 48 hours after interrupting the root stele ofPisum, wound phloem initiated (proximally or distally to the wound) to reconnect the vascular stumps was found to contain some nucleate wound-sieve elements. At the elongating end of an incomplete wound-sieve tube these elements exhibit a sequence of ultrastructural changes as known from protophloem-sieve tubes. Elongation occurs by the addition of newly divided (wound-) sieve-element/companion-cell complexes. In order to dedifferentiate and assume a new specialization formerly quiescent stelar or cortical cells require at least one (mostly more) preliminary division. Companion cells are consequently obligatory sister cells to wound-sieve elements.By reconstruction using serial sections it could be shown that wound-sieve tubes elongate bidirectionally, starting in an early activated procambial cell of the stele. The elongation is directed by the existence of plasmodesmata, preferably when lying in primary pit fields, and by the plane of preceding divisions. Thus, the developing wound-sieve tube can deviate from the damaged bundle and radiate into the cortex as soon as the plane of the preceding divisions is favourable. In the opposite direction, elongating wound-sieve tubes run parallel to pre-existing phloem traces, thus broading their base at the bundle for the deviating part of the wound-sieve tube. Frequently an individual wound-sieve tube is supplemented at the bundle by a further wound-sieve tube which is partly running parallel to it. Both sieve tubes are interlinked with sieve plates by three-poled sieve elements.Ultrastructurally, the developmental changes of nucleate wound-sieve elements follow the known pattern. In spite of its contrasting origin and odd shape a mature wound-sieve element eventually has the same contents as regular sieve elements: sieve-element plastids, mitochondria, stacked ER and small amounts of P-protein within an electronlucent cytoplasm.     

Phenogenetics of triploid intersexes in Drosophila melanogaster

Triploid intersexes homozygous for a mutant (msl-2) known to impede the hyperactivation of the X chromosome in diploid males differentiate into adults, sexually indistinguishable from their heterozygous sibs. A shift toward female sexual differentiation mediated by manipulating the rearing temperature is accompanied by an apparent increase in the level of an X-linked gene product. This unexpected result is rationalized in terms of differential lethality of individuals at the two extremities of the distribution of X-activity levels in intersexes raised at a particular temperature. No evidence of a mosaicism comparable to the sexual mosaicism exhibited could be found with respect to an X-linked gene product in triploid intersexes.     

Fluorescence microscopy of the endomembrane system of living plant cells

Abstract The fluorochrome Auramine O has been evaluated as a fluorescent probe for components of the endomembrane system of living plant cells. At 0.001% w/v the compound did not inhibit seedling growth or cytoplasmic streaming but stained the nuclear envelope, endoplasmic reticulum and Golgi apparatus. The three-dimensional, structural interrelationships of these organelles in living tissues could be resolved after minimal tissue preparation. The method is also a valuable control treatment for use in conjunction with electron microscope fixation procedures. It provides a rapid means of examining dynamic changes in the endomembrane system associated with cell development and differentiation and could have application in monitoring the effects of applied physiological or chemical stress.     

Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor

Prostate carcinomas are among the most frequently diagnosed and death causing cancers affecting males in the developed world. It has become clear that the molecular mechanisms that drive the differentiation of normal prostate cells towards neoplasia involve multiple signal transduction cascades that often overlap and interact. A critical mediator of cellular proliferation and differentiation in various cells (and cancers) is the cAMP-dependent protein kinase, also known as protein kinase A (PKA), and its activating secondary messenger, cAMP. PKA and cAMP have been shown to play critical roles in prostate carcinogenesis and are the subject of this review. In particular we will focus on the cross-talk between PKA/cAMP signaling and that of the androgen receptor.     

Genetic structure and differentiation of the Japanese extremely long-tailed chicken breed (Onagadori), associated with plumage colour variation: suggestions for its management and conservation

The Onagadori is a distinguished chicken breed that is characterized by an extremely long tail in the male. In this breed, three different plumage colour varieties have been developed (black-breasted white, black-breasted red and white) in which the black-breasted white is believed to be the original colour of the Onagadori, based on historical records. To establish a conservation strategy, 176 birds were genotyped for autosomal microsatellites. Significant genetic distinctness was found between the original (black-breasted white) and two derivative varieties ( F _ST = 0.091 and 0.093). At the same time, a Bayesian model-based clustering revealed that the majority of individuals belonging to the black-breasted red and white varieties had an extremely low proportion of the genome shared with the original type (black-breasted white). This suggests that derivative varieties were created by crossing with other breeds, with low introgression of the original-type genome. We propose that the three plumage colour varieties should be treated as separate genetic units in a conservation programme.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.