Spermatogenesis in goats with and without scrotum bipartition

The objective of the present research was to quantify the seminiferous epithelium cells, spermatogenesis efficiency and characterize the ultrastrucure of Sertoli cells in goats. Eighteen goats were used and divided into three groups: Group I - goats without bipartition of the scrotum; Group II - animals with bipartition of the scrotum in up to 50% of the testicular length; Group III - goats with bipartition of the scrotum in more than 50% of the testicular length. The goat testes in Group III had a greater number of primary spermatocytes (25.37 ± 4.55 cells per cross sections), spermatids (112 ± 15.12 cells per cross sections), and Sertoli cells (9.46 ± 1.74 cells per cross sections) than the animals in Groups I and II (P<0.05). The spermatogenic mitotic, meiotic, and general efficiency were greater in animals in Group III (1.25 ± 0.28; 5.12 ± 1.63; 6.44 ± 1.96) when compared to those in Groups I and II. Sheet-like processes originated from the Sertoli cell body as simple and smooth structures which involved almost all the surface of germ cells. Slender cord-like processes originated from Sertoli cells and also from the sheet-like processes. The relative frequency of the cycle stages showed differences among the groups of goats studied, and the highest frequency was in Stage 3 (20.68% for goats in Group I, 21.15% for those in Group II, and 16.89% for the animals in Group III). In conclusion, goats with bipartition of the scrotum have a greater number of germ and Sertoli cells per cross section of seminiferous tubule, that indicated a greater sperm production when compared to the other groups, and the ultrastructure of the Sertoli cell process did not present any relationship with bipartition of the scrotum.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Mapping the testicular interstitial fluid proteome from normal rats

Communication between the testicular somatic (Sertoli, Leydig, peritubular myoid, macrophage) and germ cell types is essential for sperm production (spermatogenesis), but the communicating factors are poorly understood. We reasoned that identification of proteins in the testicular interstitial fluid (TIF) that bathes these cells could provide a new means to explore spermatogenic function. The aim of this study was to map the proteome of TIF from normal adult rats. Low‐abundance proteins in TIF were enriched using ProteoMiner beads and identified by MALDI‐MS/MS, recognizing 276 proteins. Comparison with proteomic and genomic databases showed these proteins originated from germ cells, somatic cells (Sertoli, peritubular myoid, Leydig), and blood plasma. In silico analysis revealed homologues of >80% TIF proteins in the human plasma proteome, suggesting ready exchange between these fluids. Only 36% of TIF proteins were common with seminiferous tubule fluid that transports mature spermatids to the epididymis, indicating these two fluids are quite different. This TIF proteome provides an important new resource for the study of intercellular communication in the testis.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.     

Effects of androgen deprivation on the histology of adult chimpanzee testis

Until primate sperm are exposed to the unique microenvironment of the epididymis, they are not capable of fertilization or vigorous motility. Many of the proteins that contribute to the unique microenvironment of the primate epididymis, and thus to sperm maturation, are dependent on androgens to induce their synthesis and secretion. GnRH antagonists have proved effective in suppressing LH and testosterone synthesis and secretion, and thus in maintaining a state of androgen deprivation or functional hypogonadotropism. We report here the effects of GnRH antagonist-induced androgen-deprivation on the histology of the testicular interstitium and seminiferous epithelium of the adult male chimpanzee. After only 21 days of androgen-deprivation, chimpanzee testicular tissues exhibit specific atrophic changes, including the loss of contact between developing spermatocytes and between Sertoli cells and their developing spermatids, alterations in cell development resulting in missing maturation steps (elongating Sc and structurally complete Sd2 spermatids) and inappropriate cell associations, varying degrees of cytoplasmic degradation in germ cells, Sertoli cells, and Leydig cells, and a tubular lumen obscured by masses of sloughed primary and secondary spermatocytes and what appear histologically to be Sb1 and Sd1 spermatids.     

Effect of the microtubule disrupting agents,colchicine and vinblastine,on seminiferous tubule structure in the rat

Injections of colchicine or vinblastine were given intratesticularly and rats sacrificed 6 and 12 hr later. Colchicine and vinblastine produced identical morphological patterns of response in the seminiferous tubules resulting in arrest of germcell mitoses and meioses and a rapid depletion of the microtubules normally found within the Sertoli cell. Sloughing of cells into the lumen of seminiferous tubules was the most prominent feature noted. Germ cells and portions of the apical Sertoli cells were frequently sloughed together where they remained in close association. Usually germ cells and associated Sertoli cell fragments were cleaved from the wall of the seminiferous tubule at a level between dissimilar generations of germ cells, e.g. between spermatocytes and spermatids. This selective sloughing probably occurred as the result of the support normally provided by intercellular bridges which link clones of like germ cell types. Sequential steps in the process leading to sloughing of Sertoli-germ cell associations could be inferred from observations made in plastic 1 μm sections. Cell sloughing at 12 hr post-injection was generally more extensive. It was frequently noted that germ cells and the apical portions of Sertoli cells had been extruded to the level of the most adluminal tight junctions forming the blood-testis barrier. It was concluded that disruption of Sertoli microtubules was responsible for sloughing of Sertoli fragments and associated germ cells, and that the cytoskeletal support of the Sertoli cell was, at least in part, dependent upon the integrity of Sertoli microtubules. The Sertoli cell could not round-up after loss of its cytoskeletal support, due to the numerous attachment devices known to link it with various apically positioned germ cells. Thus, the cell was severed at some point along its delicate apical processes, as the consequence of forces produced by the ‘rounding-up’ process. Long-term sacrifice after vinblastine or colchicine treatment allowed the Sertoli cells to regain microtubules and long processes but not their typical configuration. Spermatogenesis remained severely impaired.     

The interstitial origin of germinal cells in the testis of the stickleback

The brook stickleback, Culaea inconstans (Kirtland), in common with other bony fishes, lacks a germinal epithelium in the tubules of the testis, and the tubule wall is composed of a thin, discontinuous layer of myoid cells and collagenous fibers. Labelling of germ cells with tritiated thymidine has shown that the germ cells are derived from clumps of spermatogonia in the interstitial area. Large companion cells within the lumina of the tubules extend their processes to engulf spermatogonia from the interstitium which then enter the lumen of the tubule. Subsequent development of the germ cells takes place within individual compartments formed by folds of the plasma membrane of a companion cell. The companion cell, together with its complement of germ cells, constitutes a cyst. A companion cell may surround spermatogonia in the interstitium and at the same time encompass residual sperm of the previous season within the lumen. The plasma membranes of the germ cells and the companion cells remain discrete. Mature sperm are released into the lumen of the tubule and the companion cell again extends its processes into the interstitium and engulfs more spermatogonia for the following year. Companion cells may be homologous to the Sertoli cells of higher vertebrates although their processes penetrate the interstitium during the initial stages of spermatogenesis and they do not contain a permanent stock of spermatogonia.     

Trout sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study

In order to characterize trout Sertoli cells and germ cells obtained after testis dissociation and cell separation, we have studied their morphology, ultrastructure, survival, and ability to express differentiated activities in primary cultures. After dissociation, the fine structure of Sertoli cells does not differ from that observed in situ and only minor changes are shown for at least 13 days. Until they are flattened in a monolayer, they keep the ability to retain germ cells on their surface. When flattened, some of them are able to divide. At the opposite of meiotic germ cells, spermatogonia can develop independently of Sertoli cells. They are able to proliferate during at least 10 days. Spermatocytes and spermatids are obtained as single cells and multinucleated giant cells (symplasts). In the absence of somatic cells, their maximal viability is approximately 5 days, whereas spermatocytes adhering to Sertoli cells can survive at least 10–12 days, provided trout lipoproteins are present. Spermatocytes are able to differentiate to spermatids, although this process is impaired for some ceils. The adhesion of spermatogonia and spermatocytes to Sertoli cells is specific, mediated by desmosome-like junctions and favored by lipoproteins. These data are compared to what is known in mammals and in amphibians.     

Ultrastructure of Sertoli-cell penetrating processes found in germ cells of the golden-mantled ground squirrel (spermophilus lateralis)

We have studied the ultrastructure of Sertoli-cell processes that extend into developing germ cells of the ground squirrel (Spermophilus lateralis). In other mammals it is speculated that these processes anchor germ cells to the seminiferous epithelium and transfer materials between Sertoli and germ cells. In the ground squirrel, Sertoli-cell projections first appear in round spermatids and consist of regions containing numerous mitochondria and intermediate filaments together with areas composed mainly of a fine filamentous matrix. Also present are what may be desmosomelike junctions with adjacent germ cells. During spermatogenesis, numerous changes in the penetrating processes and their internal composition occur. Especially significant are those occurring during the movement of residual cytoplasm basally over spermatid heads: some Sertoli-cell processes contain microtubules, mitochondria, and vesicular elements, but also present are regions that lack organelles and appear simply as thin lamellae of cytoplasm that line cavernous invaginations of the germ cell. Coated vesicles and pits are present in processes and adjacent germ-cell regions at all stages of spermatogenesis. Our observations are consistent with the suggestions that Sertoli-cell processes have an attachment function and that they also may facilitate the movement of residual cytoplasm into the epithelium. Further, they indicate that these structures might be involved with receptor-mediated edocytosis.     

Spermiogenesis in the bullfrog (Rana catesbeiana): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The process by which spermatid cytoplasmic volume is reduced and cytoplasm eliminated during spermiogenesis was investigated in the bullfrog Rana catesbeiana. At early phases of spermiogenesis, newly formed, rounded spermatids were found within spermatocysts. As acrosomal development, nuclear elongation, and chromatin condensation occurred, spermatid nuclei became eccentric within the cell. A cytoplasmic lobe formed from the caudal spermatid head and flagellum and extended toward the seminiferous tubule lumen. The cytoplasmic lobe underwent progressive condensation whereby most of its cytoplasm became extremely electron dense and contrasted sharply with numerous electron-translucent vesicles contained therein. At the completion of spermiogenesis, many spermatids with their highly condensed cytoplasm still attached were released from their Sertoli cell into the lumen of the seminiferous tubule. There was no evidence of the phagocytosis of residual bodies by Sertoli cells. Because spermatozoa are normally retained in the testis in winter and are not released until the following breeding season, sperm were induced to traverse the duct system with a single injection of hCG. Some spermatids remained attached to their cytoplasm during the sojourn through the testicular and kidney ducts; however, by the time the sperm reached the Wolffian duct, separation had occurred. The discarded cytoplasmic lobe (residual body) appeared to be degraded with the epithelium of the Wolffian duct. It was determined that the volume of the spermatid was reduced by 87% during spermiogenesis through a nuclear volume decrease of 76% and cytoplasmic volume decrease of 95.3%.     

Relationship of intratesticular testosterone content of stallions to age, spermatogenesis, Sertoli cell distribution and germ cell-Sertoli cell ratios

Testes were obtained from 47 1-20-year-old stallions during the natural breeding season. Total testicular testosterone and testosterone/g testis increased with age (P less than 0.005), and total testicular testosterone was associated with larger testis size (P less than 0.05). Neither testosterone per gram nor per paired testes were related to total Sertoli cell number (P greater than 0.05), but greater testosterone per paired testes was associated with fewer Sertoli cells per unit of seminiferous tubule length (P less than 0.005) or basement membrane area (P less than 0.02) and with a higher number of germ cells supported per Sertoli cell (P less than 0.05). Although values for testosterone per gram and per paired testes were unrelated (P greater than 0.10) to sperm production/g testis or to the yield of spermatids/spermatogonium, testosterone per paired testes was positively related to sperm production per paired testes (P less than 0.05). It is concluded that intratesticular testosterone increases with age, is related in a positive manner to quantitative rates of sperm production, and can account for some of the differences in sperm production among individual stallions within a single breeding season.     

Intermediate filaments in the testis of the teleost mosquito fish Gambusia affinis holbrooki: a light and electron microscope immunocytochemical study and Western blotting analysis

Summary A light and electron microscope immunocytochemical study and Western blotting analysis has been performed on intermediate filaments (vimentin, desmin and cytokeratins) in the testis of the teleost fish Gambusia affinis holbrooki. An immunoreaction to vimentin was observed in the epithelium of the efferent ducts, testicular canal and their surrounding peritubular cells. Positive vimentin immunostaining was also observed in the cells located around seminiferous tubules (boundary cells), Leydig cells, interstitial fibroblasts, chromatophores, and blood vessel endothelial cells. In contrast to mammals, no vimentin immunoreactivity was found in the Sertoli cells. Immunoreactivity to desmin was weak in the epithelial cells of the efferent ducts and testicular canal and intense in the peritubular cells that surrounded these ducts. Desmin immunoreactivity was also observed in the seminiferous tubule boundary cells. The immunoreactivity was weak in the boundary cells that surrounded germ cell cysts containing spermatogonia or spermatocytes and intense in the boundary cells around cysts with elongated or mature spermatids. Immunoreactivity towards cytokeratins was observed only in testicular blood vessels. Cytokeratin immunolabelling was intense in the endothelium and weak in the vascular smooth muscle cells. No cytokeratin immunoreactivity was found in the Sertoli cells, germ cells, interstitial cells or in the efferent duct epithelium. The absence of intermediate filaments in the Sertoli cells, the absence of cytokeratins in the epithelium of the sperm excretory ducts, and the presence of desmin filaments in these epithelial cells are the most important differences with regards to the intermediate filament phenotype in mammalian testes.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca)

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by “A” spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.     

The Sertoli cell in vivo and in vitro

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

Early stages of testicular differentiation in the rat

Summary The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells show an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords.The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.     

A light and electron microscopic study on the organization of the testis and the semicystic spermatogenesis of the genus Scorpaena (Teleostei,Scorpaenidae)

The testicular organization and semicystic spermatogenesis of Scorpaena porcus and Scorpaena scrofa are analyzed by means of optic and electron microscopy and immunohistochemical techniques. The testicular structure of S. porcus and S. scrofa belongs to the unrestricted spermatogonial type, but has typical features of the restricted type. Moreover, the structure presents an epithelioid arrangement of Sertoli and germ cells rather than the germinal epithelium that appears in the majority of teleosts. After the cysts open, Sertoli cells hypertrophy and remain on the basement membrane, linked by interdigitations and tight junctions and bordering the lumen of the lobule, which at this moment works as an efferent duct. Secretions of Sertoli cells usually function in the nutrition of germ cells, and they seem to contribute in it even in this kind of spermatogenesis in which the free lumen spermatids do not have any connection with Sertoli cells. In addition, Sertoli cells can divide after the cysts have broken apart and hypertrophied, suggesting that they are still important for the final maturation of spermatozoa and seminal fluid formation. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.