In vivo analysis of phagocytosis of apoptotic cells by testicular Sertoli cells

Sertoli cells, a somatic cell type present within the seminiferous tubules of testes, are responsible for the phagocytic elimination of apoptotic spermatogenic cells. We here established an in vivo assay system that enables us to quantitatively analyze Sertoli cell phagocytosis of apoptotic cells in testes of live mice. Apoptotic cells were injected into the seminiferous tubules of spermatogenic cell-depleted mice, and the occurrence of phagocytosis by Sertoli cells was examined by histochemically analyzing testis sections or dispersed testicular cells. We reproducibly observed similar levels of phagocytosis in either examination, and the ratio of Sertoli cells that engulfed injected apoptotic cells was almost the same between the two examinations. These results indicated that a quantitative in vivo assay system was established using the seminiferous tubules of live mice as 'test tubes.' We then determined the requirements for Sertoli cell phagocytosis of apoptotic cells using this assay. For this purpose, apoptotic cells were injected together with various phagocytosis inhibitors, and the extent of phagocytosis by Sertoli cells was determined. The results revealed that Sertoli cells phagocytose apoptotic cells in a manner dependent on class B scavenger receptor type I (SR-BI) of Sertoli cells and phosphatidylserine exposed at the surface of target cells, as previously observed in vitro using primary cultures of dispersed rat testicular cells. Furthermore, the amount of SR-BI in Sertoli cells increased after injection of apoptotic cells into the seminiferous tubules, suggesting a positive feedback regulation of the expression of this phagocytosis receptor.     

Seminiferous tubule involution in elderly men

The observation of different types of seminiferous tubules (from tubules with normal spermatogenesis to sclerosed tubules) in aging human testes points to the progressive stages of tubular involution in elderly men. The tubules with hypospermatogonesis (reduced number of elongated spermatids) show numerous morphological anomalies in the germ cells, including multinucleated cells. Abnormal germ cells degenerate, causing Steroli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytized by Sertoli cells lead to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with spermatids, but progressively affects the preceding germ cell types, and tubules with maturation arrested at the level of spermatocytes or spermatogonia are observed. Simultaneously, an enlargement of the tunica propria occurs. This leads to the formation of sclerosed tubules, some of which display a low seminiferous epithelium consisting of a few cells--including lipid-loaded Sertoli cells and both Ap and Ad spermatogonia--and others, showing complete sclerosis, are devoid of seminiferous epithelium. The development of tubular involution is similar to that reported after experimental ischemia, which also seems to cause nonspecific effects on the testis such as multinucleate cells, vacuoles, and increased lipids in Sertoli cells.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

Content of K+ and Na+ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes

Seminiferous tubules of rats exposed to x-irradiation before birth were subjected to micropuncture in situ at 50 days of age to obtain samples of fluid 4 h after ligation of efferent ducts. The concentrations of cations in this fluid were: potassium, 39.7 +/- 1.2 mM, and sodium, 136.3 +/- 1.2 mM (means and standard errors, n = 5). Histologic examination revealed that germ cells constitute less than 1% of the cell population within the seminiferous tubules of these rats; the remaining cells were all Sertoli cells. Sertoli cells showed efflux of 86Rb+ with t1/2 of approximately 11 min and an active ATPase in plasma membranes. These activities were similar to those of Sertoli cells from normal rats. Germ cells from normal rats showed less rapid efflux of 86Rb+ (t1/2 greater than 60 min) and less active Na+/K+ ATPase in plasma membranes. It is concluded that Sertoli cells are responsible for the high concentration of potassium in seminiferous tubule fluid and that plasma membranes of these cells contain an active K+ pump that is not inhibited by ouabain (1 mM).     

Fine structural changes in Sertoli and Leydig cells during the reproductive cycle of the ground squirrel, Citellus lateralis

During spermatogenesis in sexually mature ground squirrels Leydig and Sertoli cells were morphologically well differentiated. For Leydig cells the most prominent organelles were lipid droplets, mitochondria with tubulo-vesicular cristae and abundant agranular reticulum organized as a mass of anastomosing tubules. These morphological criteria suggest that the Leydig cells were steroidogenically active. Sertoli cells exhibited a topographical distribution of certain organelles with basal regions containing stacks of granular reticulum, and large areas of agranular reticulum. The cytoplasm surrounding maturing germ cells contained numerous microtubules, and an adluminal layer of spermatids at a certain stage of spermiogenesis became enveloped by Sertoli cytoplasm containing an enormous proliferation of agranular reticulum. The presence of these organelles in Sertoli cells suggests that during spermatogenesis they are active in the synthesis of proteins and steroids. In particular the mass of agranular reticulum surrounding late stage spermatids indicates that steroids may be required for spermatid maturation and/or spermiation. By contrast Leydig and Sertoli cells observed during testicular regression, when only spermatogonia remain in the seminiferous tubules, had undergone structural changes. Leydig cells were still numerous and large with abundant agranular reticulum that was now organized as a loose assemblage of single unbranched tubules. Sertoli cells were drastically reduced in both cytoplasmic volume and content of organelles.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

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.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Functional changes of mice Sertoli cells induced by Cr(V)

Transport of macromolecules from the interstitial testis tissue to cells at the adlumenal compartment of the seminiferous epithelium occurs naturally through Sertoli cells. In previous studies we have shown that Cr(V) intoxication disturbed spermatogenesis in mice. To test if Sertoli cells are affected by chromium, a well proved carcinogen, the uptake and the horseradish peroxidase transport ability of isolated seminiferous tubules of mice administered with a chromium(V) compound, have been studied. Male CD-R mice were exposed daily for 5 days to [Cr^V-BT]^2– through subcutaneous injection and comparisons were made with groups of vehicle-treated mice. Using an in vitro assay we demonstrated that the seminiferous tubules were able to uptake and transport the tracer, in a much faster way than controls, mainly via intercellular and transcellular pathways, providing evidence that this functional role of Sertoli cells is affected by the Cr(V) compound. These findings might improve the knowledge on the toxicity mechanisms of chromium.     

Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters.

The distribution of Mg++-activated ATPase was determined with light and electron microscopy in normal and degenerating seminferous tubules. In the normal animals ATPase was localized in the interface between spermatids and Sertoli cells, in association with the cytoplasmic filaments contained within Sertoli cell processes, and in the lymphatic endothelium. ATPase activity increased in degenerating tubules as observed by light microscopy. Electron microscopic investigations of the degenerating tubules which contained only spermatogonia and Sertoli cells revealed reaction product on the outer surface of the Sertoli cell processes and within the interface between adjacent Sertoli cells. Reactaction product was also observed in the Sertoli cell processes between the cytoplasmic filaments and the cell membrane. Where filaments were absent in Sertoli cell processes, no reaction product was observed. These electron microscopic studies indicate that the increase in ATPase activity in testicular degeneration is probably a relative increase due to a loss of the germinal elements of the tubular epithelium and subsequent apposition of the Sertoli cell processes. We speculate that the ATPase activity localized within the Sertoli cell processes may be involved in providing an energy source for filament motility.     

Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli–Sertoli cell junction and Sertoli–spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.     

Sertoli cell development and function in an animal model of testicular dysgenesis syndrome

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.     

The double nucleus of the sertoli cell in the lizard Anolis carolinensis

Over 90% of the Sertoli cells in the testes of adult lizards (Anolis carolinensis) are binucleate. The nuclei occur in closely associated pairs in the basal cytoplasm of the Sertoli cells that line the testis tubules. The two nuclei of a pair are of similar volume, and each usually contains one conspicuous rounded nucleolus. The average volume of individual nuclei varies from 367.8 mum(3) in spermatogenically active testes in March to 172.5 mum(3) in September, when testes are regressed. The irregular shape of the Sertoli nuclei is particularly pronounced during testicular regression. Until initiation of spermatogenesis in hatchling lizards, Sertoli cells have a single nucleus containing patches of hetcrochromatin. With the appearance of prophase stages of primary spermatocytes, a few paired Sertoli nuclei can be found, and the nuclei increasingly exhibit the homogeneous euchromatic nucleoplasm of the adult. The average volume of individual nuclei in lizards under 4 months of age is less than a third the volume of Sertoli nuclei in reproductivcly active adults. The appearance of binucleate cells at this time documents a doubling of the amount of desoxynucleic acid in Sertoli cells preparatory to their growth and expanded functions during spermatogenesis.     

Cyclic localization change of Golgi apparatus in Sertoli cells induced by mature spermatids in rats

Previously we reported that the intracellular localization of the Golgi apparatus of rat Sertoli cells changes during the seminiferous epithelial cycle, and that the cyclic changes seem to be correlated to specific generations of germ cells. To ascertain which generations of germ cells are responsible for the cyclic changes, we determined the relative volume of the Golgi apparatus within the basal, mid, and apical cytoplasm of Sertoli cells in testes with and without mature spermatids. In normal adult rats, the Golgi apparatus was usually localized exclusively in the basal cytoplasm, whereas at stages VII-IX it increased remarkably in mid and apical cytoplasm, with a concomitant decrease in the basal cytoplasm. In young adult testes without spermatids at steps 15-19 of spermiogenesis (2nd layer spermatids), the Golgi apparatus was localized in the basal cytoplasm throughout the seminiferous epithelial cycle. Orchiopexy maintained for 35 days following 60 days of cryptorchidism allowed germ cells to regenerate to spermatids at steps 1-14 of sperminogenesis (1st layer spermatids), but failed to change the intracellular localization of the Golgi apparatus in Sertoli cells. At 50 days after orchiopexy, when all generations of germ cells appeared in the tubules, the cyclic changes in localization of the Golgi apparatus were restored similar to those in normal adult testes. These findings indicate that the cyclic change in localization of the Golgi apparatus in Sertoli cells is evoked by the presence of 2nd layer spermatids.     

Response to cryptorchidism of the testis and epididymis of the opossum (Didelphis virginiana).

Adult male opossums, Didelphis virginiana, were rendered hemicryptorchid for 35 days. The cyrptorchid testis exhibited a significant reduction in weight, while the contralateral testis had a compensatory weight gain compared with testes of untreated animals. Histological changes in the cryptorchid testis included fibrosis of the tunica propria, involution of the seminiferous tubules and an apparent increase in the interstitial tissue. Many seminiferous tubules were empty and germinal cells were absent. Some Sertoli cells persisted, but the cytoplasm was vacuolated. Cryptorchid testes were characterized by mononuclear leucocytic invasion around the tubules, and some eosinophils were observed. Cryptorchidism in the opossum may induce a reaction similar to experimental orchitis.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.