Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in R. norvegicus

Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and non-targeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either Western Blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein–related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were “less distinct” than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin related junctions (ESs).

Cyclooxygenase and prostaglandins in somatic cell populations of the testis

Prostaglandins (PGs) are synthesized through the action of the rate-limiting enzyme cyclooxygenase (COX) and further specific enzymes. The development ofCox-deficient mice in the 1990s gave insights into the reproductive roles of PGs. FemaleCox-knockout mice were subfertile or infertile. Interestingly, fertility was not affected in male mice deficient inCox, suggesting that PGs may not be critical for the functioning of the testis. However, this conclusion has recently been challenged by observations of important roles for PGs in both physiological and pathological processes in the testis. The two key somatic cell types in the testis, Leydig and Sertoli cells, express the inducible isoenzyme COX2 and produce PGs. Testicular COX2 expression in these somatic cells is regulated by hormonal input (FSH, prolactin (PRL), and testosterone) as well as by IL1β. PGs modulate steroidogenesis in Leydig cells and glucose uptake in Sertoli cells. Hence, the COX2/PG system in Leydig and Sertoli cells acts as a local modulator of testicular activity, and consequently may regulate spermatogenic efficiency. In addition to its expression in Leydig and Sertoli cells, COX2 has been detected in the seminiferous tubule wall, and in testicular macrophages and mast cells of infertile patients. These observations highlight the possible relevance of PGs in testicular inflammation associated with idiopathic infertility. Collectively, these data indicate that the COX2/PG system plays crucial roles not only in testicular physiology (i.e., development, steroidogenesis, and spermatogenesis), but more importantly in the pathogenesis or maintenance of infertility status in the male gonad. Further studies of these actions could lead to new therapeutic approaches to idiopathic male infertility.Free German abstractA German translation of this abstract is freely available athttp://www.reproduction-online.org/content/149/4/R169/suppl/DC1.Free Spanish abstractA Spanish translation of this abstract is freely available athttp://www.reproduction-online.org/content/149/4/R169/suppl/DC2.

Ultrastructure of Renal Tubular Epithelial Cells Of Rat’s Kidneys after Administration of L-Arginine

Sixteen white Wistar female rats were divided into two equal groups. Experimental group receivedper os40 mg/kg b.w. of L-arginine, every other day for 2 weeks and were decapitated after 3 weeks of the experiment. Control rats received in the same manner 2 ml of distilled water and were decapitated after 3 weeks of the experiment. The renal lesions observed under electron microscope were of focal character and concerned only the experimental group. The tubules with necrotic cells were observed among normal tubules or single normal epithelial cells of the tubular wall. The boundaries between epithelial cells of the tubule wall were blurred. The mitochondria indicated abnormal structure. Numerous lysosomes and peroxysomes with dark, homogenous content were observed. The rough endoplasmic reticulum had widened channels and was focally completely destroyed. The nucleus of damaged cells was most commonly located in one of the cell poles; its shape was changed and visibly smaller than the nuclei of normal cells. Condensation and peripherally located chromatin were noticed. The lesions observed were characteristic for apoptotic cells.

Bartter syndrome in homozygous twins

Bartter syndrome is characterized by hyperreninemia against the background of hyperplasia of juxtaglomerular cells, hyperaldosteronuria, potassium deficiency, metabolic alkalosis, and vascular resistance to angiotensin.A study of the pathogenetic aspects of this disease allowed a number of authors suppose that this syndrome is associated with autosomal recessive inheritance, in which there may be no sensitivity of vessels to the pressor effect of angiotensin II, impaired renal reabsorption of sodium and chlorine, increased renal production of prostaglandin E. Primary renal defect, leading to the loss of potassium also is not excluded, because bilateral adrenalectomy does not completely reduce hypokalemia. However, it is not always possible to establish a primary genetic defect.The clinical symptoms of this disease are characterized by adynamia, polyuria, polydipsia, headache, and sometimes vomiting. Blood pressure is normal or even low. Children have a lag in mental and physical development.The pathophysiology of clinical symptoms is caused either by a hereditary disorder of tubular reabsorption of potassium and secondary sodium due to an enzymatic defect in the tubule wall, or by resistance of target cells to the stimulation by renin, angiotensin, and aldosterone, the production of which increases due to a disturbed feedback mechanism.In foreign literature, a description of this syndrome is quite rare. This fact was the basis for the publication of a similar clinical syndrome in homozygous twins - brothers A. and D. born in 1967, who were followed up for 3 years.

Ultrastructure of the malpighian tubules in adult female Dermacentor variabilis

The excretory system of the American dog tick Dermacentor variabilis (Say) consists of a pair of Malpighian tubules that end in a blind ampulla and insert into the rectal sac which in turn leads to the anus. Visceral muscle envelopes the tubules. The single layer of epithelial cells in the tubule wall have an ultrastructure characteristic of cells that transport fluids and ions, e.g. an infolded basal plasma membrane, relatively large numbers of mitochondria, and an extensive development of microvilli on the apical luminal surface. Ixodid ticks have a cycle of excretion during their life span as an adult. Most excretory activity occurs during and after feeding. The purpose of this study was to describe the ultrastructure of the Malpighian tubules in an unfed tick, a fed tick and an ovipositing tick, in an attempt to correlate ultrastructure with excretory activity. We subdivided the tubules into distal, middle and proximal regions in which each area was about 1/3 of a tubule. The proximal region was closest to the rectal sac.

The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study

The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

Effects of luminal fluid anions on calcium transport by proximal tubule

To determine whether the anion composition of tubule fluid affects calcium absorption by the renal proximal tubule, in vivo microperfusion techniques were employed in anesthetized rats. Experiments were designed so that total calcium and sodium concentrations were kept constant in fluids entering the tubule. A control solution, in which the main anion was chloride, was modified either by addition of ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetra-acetic acid or by replacing most of the chloride with nitrate, thiocyanate, sulfate, or citrate. Sufficient mannitol was added to the perfusion fluids to reduce net fluid flux to near zero. Net fluxes of calcium and sodium were calculated from measurements of total concentrations in perfused and collected fluids. Electrochemical driving forces across the tubule wall were calculated from measurements of transepithelial voltage and of Ca2+ activity in perfused and collected fluids. Results showed that calcium absorption by the proximal tubule depends on both the luminal Ca2+ activity and the transepithelial voltage. With zero transepithelial electrochemical driving force calcium absorption was significantly different from zero. Calcium and sodium transport rates were seen to vary independently. We conclude that the calcium absorptive mechanism involves active transport and can be dissociated from the sodium transport pathway. Calcium transport is also affected by changes in transepithelial electrochemical driving forces with an apparent permeability similar to values reported for sodium and potassium.

Fluid reabsorption by Necturus proximal tubule perfused with solutions of normal and reduced osmolarity

Fluid absorption in Necturus proximal tubule was studied when the kidneys were perfused with solutions of different osmolarities. The rate of fluid absorption was inversely proportional to the perfusion fluid osmolarity, while Na uptake remained constant. No difference was detected between the collected and injected luminal fluid, i. e. reabsorption was isotonic at normal and reduced osmolarities. The transtubular osmotic permeability remained fairly constant under the different per­fusion osmolarities. Using our experimental results to test various models based on osmotic equilibration across the tubule wall we show that none of these provides an adequate mechanism for fluid absorption in this epithelium.