Evaluation of risk of symptomatic cytomegalovirus reactivation in myeloma patients treated with tandem autologous stem cell transplantation and novel agents: a single-institution study.

The introduction of proteasome inhibitors and/or immunomodulators in the treatment of myeloma has led to an increase in viral infections, particularly in the Herpesviridae family. Previous studies about the risk of cytomegalovirus (CMV) reactivation after autologous stem cell transplantation (ASCT) have examined the clinical outcome after the first ASCT; however, only 1 study to date has investigated the risk of CMV reactivation after a second transplantation. To address this issue, we performed a retrospective chart review on 78 consecutive myeloma patients (median age 56 years) who underwent a tandem non-CD34(+) selected ASCT after induction treatment with either conventional chemotherapy (n = 42) or with novel agents (n = 36), respectively. All subjects had been mobilized and conditioned with cyclophosphamide plus granulocyte colony-stimulating factor and melphalan alone, respectively. CMV DNA load in the blood has been determined by polymerase chain reaction in the case of a clinical suspicion of CMV reactivation; therefore, routine monitoring was not performed. Considering the outcome of both the first and the second transplantations, we observed a total of 13 episodes of symptomatic CMV reactivation (13/156, 8%), in 12 subjects (12/78, 15%), all successfully treated. Eight subjects experienced a CMV reactivation after the first ASCT (8/78, 10%); however, only 1 of them (1/8, 12%) experienced a CMV reactivation after the second transplantation. Conversely, 4 CMV reactivations (6%) were observed after the second transplantation in the group of 70 patients who did not experience a CMV reactivation after the first ASCT. No statistically significant difference was observed between first and second ASCT (8/78, 10% vs. 5/78, 6%; P = 0.767). Univariate analysis showed that a pre-transplant treatment with novel agents was the only baseline factor significantly associated with the occurrence of post-ASCT CMV symptomatic reactivation after the first transplant (odds ratio [OR]: 9.897; 95% confidence interval [CI]: 1.154-84.840; P = 0.021) but not after the second transplant (OR: 5.125; 95% CI: 0.546-48.119; P = 0.115). No end-organ disease or primary infection was documented. Our data suggest that second transplantation does not increase the risk of CMV reactivation in our patient population, when compared with the first one, and confirm the role of a pre-transplant treatment with novel agents as a risk factor for CMV symptomatic reactivation.

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.

FLIP is expressed in mouse testis and protects germ cells from apoptosis.

Apoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIP(L)) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIP(L) expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIP(L) expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIP(L) expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIP(L) expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIP(L), are sensitive to Fas-mediated apoptosis. These data indicate for the first time that c-FLIP(L) might control germ cell apoptosis and caspase activity in the adult testis.

Platelet-derived growth factor-BB stimulates hypertrophy of peritubular smooth muscle cells from rat testis in primary cultures.

The tunica propria of seminiferous tubules contains a particular type of smooth muscle cell (myoid cells) arranged in a contractile epithelioid layer that is responsible for sperm and tubular fluid flow. Unlike other types of smooth muscle (SM) cells, highly purified populations of peritubular smooth muscle cells (PSMC) survive and maintain their contractile phenotype in primary cultures in controlled conditions. We used this culture model to investigate the response of the SM contractile phenotype to prolonged exposure to platelet-derived growth factor (PDGF), one of the main factors involved in vascular SM pathologies. We observed that 4-day continuous exposure of PSMC to PDGF-BB at nanomolar concentrations in plain medium enhances contractile phenotype traits and induces cell hypertrophy without inducing proliferation. In Northern and Western blotting experiments, SM-alpha-actin transcript and protein were found to be markedly increased in the PDGF-BB-treated samples, which is in line with the formation of conspicuous SM-alpha-actin-containing stress fibers. Moreover, binding sites for endothelin-1 were increased, and the calcium response to the contractile agonist, determined in single fura-2-loaded cells, was enhanced. In response to PDGF-BB, the cells underwent immediate, transient contraction, as seen in a scanning electron microscope, followed by a gradual increase in size, as evaluated by cytofluorometry, and enhancement of protein synthesis. The observed pattern of response to PDGF-BB was not accompanied by cell proliferation, as assessed by [3H]thymidine incorporation and direct cell counts. Unlike other SM cell types, in which proliferation and loss of contractile traits are induced by PDGF, chronic treatment of PSMC with this growth factor results in hypertrophy rather than hyperplasia.

Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway.

The presence of endothelin (ET), a vasoconstrictor peptide, in the testis suggests that it may regulate nonvascular target cells. We investigated binding ability, regulation of inositol phosphate metabolism, changes in cytosolic free Ca2+ concentrations ([Ca2+]i), and induction of morphological changes by ET-1 in rat primary testicular myoid cell cultures. ET-1 specifically bound to highly purified rat testicular myoid cells in a time- and temperature-dependent manner. Scatchard analysis of the binding studies indicated the presence of a single class of high affinity binding sites, with an apparent Kd of 3 x 10(-10) M and a maximal binding capacity of 10(5) sites/cell. ET-1 induced both rapid production of inositol triphosphate and mobilization of [Ca2+]i in a concentration-dependent fashion. By contrast, inositol lipid metabolism was slightly affected by ET-1 in the total peritubular cell population. Purified Sertoli cells failed to show either ET-1 binding or ET-1-induced phosphatidylinositol hydrolysis. Mobilization of [Ca2+]i mostly depended upon the release of Ca2+ from thapsigargin-sensitive intracellular Ca2+, whereas it was not affected by abolishment of the Ca2+ gradient through the plasma membrane or by inhibition of L-type voltage-sensitive Ca(2+)-channels by nifedipine. These findings together with the fact that Sertoli cells are unable to respond to and bind ET-1 indicate that ET is a specific agonist of myoid cells in the seminiferous tubule and suggest a role for ET-1 in the autocrine/paracrine regulation of testicular function.

Contractile response of peritubular myoid cells to prostaglandin F2alpha.

Prostaglandin (PG) F2alpha, a well known agonist of smooth muscle, is produced in the male gonad. We have investigated whether PG F2alpha stimulates seminiferous tubule contractility through direct action on peritubular myoid cells. Myoid cells from prepubertal rats were highly purified through Percoll density gradient and cultured in vitro. Stimulation with PG F2alpha was observed to induce: (i) rapid and dose-dependent production of inositol phosphates; (ii) mobilization of Ca2+ from intracellular stores and (iii) cell contraction. Moreover, at a concentration of 10 microM the agonist was found to induce immediate contractile response of peritubular tissue in freshly explanted tubular fragments from both young and adult rats; the explants were examined in whole-mount preparations and the peritubular myoid cell layer was identified by selective staining for alkaline phosphatase activity. Our observations demonstrate that myoid cells are a direct target for PG F2alpha and suggest a role of the eicosanoid in the intragonadal control of seminiferous tubule contractility.

The saccus vasculosus of Anguilla anguilla (L.) from larva to adult. I. Ultrastructural modifications of the coronet cells.

The ultrastructure of coronet cells of the saccus vasculosus has been studied in specimens of Anguilla anguilla (L.) at different stages of its life cycle. At all the stages observed coronet cells are composed of a basal and an apical part, the latter bearing globules with primary vesicles. In the larva (a marine form) and in the fully metamorphosed small eel at the time of entry into freshwater the narrow lumen and the vesicles within the apical globules are filled with electron-dense material. In forms in which adaptation to freshwater has occurred, the saccus lumen appears expanded, the apical globules are better developed, and the electron-dense material has disappeared. It is suggested that the two situations observed represent different functional states of the organ, in relation to different conditions of environmental salinity.

Influence of Sertoli cell products upon the in vitro survival of isolated spermatocytes and spermatids.

We have investigated the effect of Sertoli cell products upon the in vitro survival of isolated pachytene spermatocytes and round spermatids using co-cultures without contact between the somatic and germ cells. 3H-leucine incorporation was measured in such conditions at different culture times and compared with that of isolated germ cells incubated in presence of different energetic substrates. Dependence upon pyruvate and Sertoli cell products with respect to lactate is shown and discussed for both cell types; differences in pyruvate requirement are reported between meiotic and haploid cells.

Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue.

We have studied the distribution of histochemically detectable alkaline phosphatase in cultures of seminiferous tubule fragments and of peritubular cells from prepubertal rats. The same material also was immunohistochemically evaluated for the presence of desmin-containing intermediate filaments. The comparative analysis of alkaline phosphatase and desmin positivity shows that alkaline phosphatase histochemistry selectively detects desmin-containing contractile cells in tubular and peritubular cell cultures. We propose alkaline phosphatase as a novel marker for myoid cells that can be of help in screening, defining, and eventually standardizing the exact composition of peritubular cell cultures, a model that is of increasing interest in the study of cellular interactions in the testis.

Early programming of maturation competence in mouse oogenesis.

Growing mouse oocytes incompetent to mature were freed of attached granulosa cells at different stages of growth, and cultured in vitro in the presence of fibroblast monolayers and/or their products. In these culture conditions, although growth was arrested, isolated oocytes survived in vitro for several days, and finally resumed meiosis spontaneously, progressing up to metaphase I. The culture time length needed for in vitro acquisition of the capacity to mature was inversely related to the initial oocyte size at the time of isolation from granulosa cells, and closely corresponded to developmental timing of acquisition of such ability in vivo. We conclude that the acquisition of mouse oocyte competence to mature follows a definite time program, which is independent of the presence of granulosa cells and of heterologous cell contacts, at least within the developmental stages studied.

Dual control of seminiferous tubule contractility mediated by ETA and ETB endothelin receptor subtypes.

Testicular myoid cells surrounding the seminiferous tubule are contractile cells responsible for peritubular contractility and for the propulsion of tubular fluid and spermatozoa. We have investigated the contractile response of rat myoid cells to endothelins (ETs) in cell and organ culture and analyzed the cell signaling involved. ET-2, ET-3, and IRL 1620, a highly selective agonist of ETB receptor, elicit [Ca2+]i increases, though with dissimilar potencies and kinetics. Competition binding assays using [125I]ET-1, [125I]ET-3 and [125I]IRL 1620 show that myoid cells express both ETA and ETB receptors with high affinity for ET-1 and ET-1/ ET-3, respectively. All endothelin isoforms activate phosphoinositide (PI) turnover, but only stimulation of the ETA receptor mediates both PI turnover and mobilization of [Ca2+]i. Although stimulation of the ETB receptor with IRL 1620 fails to produce a significant effect on inositol phosphate (IP) production, it induces mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in the absence of any measurable IP production. We also studied the effect of U-73122 [1-(6-[17-beta-3-methoxyestra-1,3,5 (10)-trien-17-yl] amino/hexyl)-1H-pirrole-2,5-dione] and its inactive analog, U-73343, on Ca2+ mobilization and IP production after selective stimulation of ET receptors. U-73122 (1 microM) completely inhibited the effect of ETA-mediated ET-1 stimulation of IP production, whereas U-73343 was inactive. However, in the presence of U-73122, the selective stimulation of ETB receptors induced the mobilization of a thapsigargin-sensitive and inositol phosphate-independent intracellular Ca2+ pool. The ETB receptor-dependent mobilization of [Ca2+]i resulted mainly from Ca2+ release from intracellular Ca2+ stores. This paper illustrates contraction of myoid cells in the seminiferous tubule in response to selective activation of either ET receptor. Scanning electron microscopy of the peritubular tissue demonstrates that the contractile response to ET was inhibited by a combination of BQ-123 and BQ-788, but not by either antagonist alone. Moreover, the observation that selective stimulation of ETB receptor with IRL 1620 also resulted in cell contraction strongly suggests that stimulation of either ETA or ETB receptors alone may be sufficient to elicit seminiferous tubule contractility. Two types of receptors account for the actions of endothelin on contractile activity of seminiferous tubule: 1) an ETA receptor that is positively coupled to phospholipase C (PLC) and Ca2+ mobilization; and 2) an ETB receptor that induces the mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in a manner independent of the formation of inositol phosphates. ET may play a complex role in regulating the flux of spermatozoa along the seminiferous tubule through its contractile effect on peritubular myoid cells.

Integrin receptor alpha 6 beta 1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium.

With the aim of investigating the presence and role of integrin receptors in cell-to-cell interactions during spermatogenesis, we have immunolocalized alpha 6A integrin chain in the rat seminiferous epithelium. In both prepubertal and adult seminiferous epithelium, the antigen was found to be restricted to definite sites of intercellular contact, following a stage-specific distribution almost invariably identical to that known for beta 1 chain. In the adult seminiferous epithelium, positivity for both antigens was found exclusively around the profiles of elongating and maturing spermatids and, in most but not all stages, at a characteristic suprabasal position. In the prepubertal rat, the antigen is localized along a very regular suprabasal line of intercellular contacts. In immunoprecipitation experiments on both seminiferous epithelium explants and Sertoli cell cultures from 3-wk-old rats, anti-alpha 6A antibody coprecipitates a polypeptide of 118 kDa, presumably corresponding to beta 1 chain. These data strongly suggest that the integrin heterodimer alpha 6A beta 1 is expressed at sites of intercellular contact in the rat seminiferous epithelium. The stage-specific and restricted pattern observed by immunofluorescence suggests that this integrin is involved in regulatory interactions between Sertoli cells and germ cells during spermatogenesis.

Direct visualization of rat peritubular myoid cell contraction in response to endothelin.

Seminiferous tubule contractility is fundamental for sperm progression towards the rete testis; hence its regulation represents a key point in male fertility. Endothelin-1 (ET-1), a potent stimulator of smooth muscle contractility, has recently been shown to be produced in the testis, as well as to bind to specific receptors on myoid cells and thereby activate intracellular signaling. The present paper illustrates contraction of isolated myoid cells in response to ET-1 both in phase-contrast and scanning electron microscopy. Moreover, a simple method is described that allows visualization of a dramatic endothelin-induced rearrangement of myoid cell actin filaments in whole-mount preparations of seminiferous tubules. The response, which can be observed within seconds from stimulation, was paralleled by cell shape changes that were well apparent in scanning electron microscopy. The present report provides the first direct evidence that endothelin is an agonist of myoid cell contractility. Moreover, the experimental approach described could represent a promising tool for the screening of potential agonists of peritubular contractility and for the analysis of their mechanisms of action. In this regard, preliminary evidence is presented on myoid cell contractile response to [Arg8]-vasopressin.

Distribution of beta 1 integrin subunit in rat seminiferous epithelium.

We have studied the presence and distribution of beta 1 integrins in the seminiferous epithelium of prepubertal and adult rats. Our immunofluorescence data show that in the adult the antibody recognizes specific areas localized around the heads of elongating and maturing spermatids and above spermatogonia at stages I-VII. The following were found to be negative: a) areas adjacent to spermatogonia at stages IX-XIV and adjacent to spermatocytes and to round spermatids; b) spermiated spermatozoa. In the prepubertal rat, positive tubules are first apparent around Day 17 of age. Immunofluorescence and immunoprecipitation studies show that Sertoli cell monolayers from 3-wk-old rats express beta integrins in vitro.

Junctional contacts between Sertoli cells in normal and aspermatogenic rat seminiferous epithelium contain alpha6beta1 integrins, and their formation is controlled by follicle-stimulating hormone.

The distribution of alpha6beta1 integrins at the level of cell-to-cell contacts within the rat seminiferous epithelium was investigated. Double fluorescence experiments using phalloidin staining of actin filaments and anti-integrin subunit antibodies showed that the receptor belongs to the Sertoli cell lateral domains engaged in the characteristic junctional structures known as ectoplasmic specializations (ES), at the level both of inter-Sertoli junctions and of the contacts between Sertoli cells and elongating spermatids. In the seminiferous epithelium of aspermatogenic testes, obtained through X-irradiation in utero (Sertoli-cell-only testes), at the level of inter-Sertoli junctions both ES and alpha6beta1 integrins are present. In order to study the dependence of alpha6beta1 receptors and ES formation upon FSH stimulation during development, 9-day-old testes were grown in organ culture in basal as well as FSH-supplemented conditions. FSH stimulation, which is necessary for the progression of spermatogenesis to early meiotic stages, appears to be required for the development of inter-Sertoli junctional structures containing ES and alpha6beta1 integrins. These observations indicate that the receptor belongs to the inter-Sertoli junctional machinery and that its expression at that level is not dependent on active spermatogenesis but requires FSH stimulation.