Cryptorchid Rats Research Articles

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.

Cloning of Novel Temperature-Related Expressed Sequence Tags in Rat Testis during Spermatogenesis

Spermatogenesis needs the relatively cool environment of the scrotum in most mammals, it would be arrested when the testis was exposed to abdominal temperature. In this study, we have used a differential display PCR technique (DD-PCR) to screen temperature-related ESTs during spermatogenesis (TRS) in scrotal testes through a unilateral cryptorchid rat model afterin situanalysis of testis cell DNA fragmentation. We reported here the cloning and sequencing of three such ESTs: TRS1, TRS3, and TRS4. Northern blot analysis confirmed that they were expressed specifically in scrotal testes.In situhybridization showed that TRS1 was mainly expressed in the spermatocytes and the round spermatids in scrotal testis. Homology searches revealed that TRS1 and TRS3 were unknown cDNA sequences, and TRS4 was identical to a known EST whose function had not been reported. TRS1, TRS2, and TRS3 were first found to be temperature-related during spermatogenesis.

Experimental cryptorchidism induces a change in the pattern of expression of LH receptor mRNA in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression.

Fertility and histological studies of the contralateral testes in two different intra- and extra-abdominal rat models of unilateral cryptorchidism.

To investigate the effect of location of the testis on both testicular development and fertility in unilateral cryptorchidism. Forty-nine Wistar King A newborn male rats were divided into three groups. In group 1 (15 rats) intra-abdominal unilateral cryptorchidism were created by the intra-abdominal fixation of the testis in the neonatal period. In group 2 (16 rats) extra-abdominal unilateral cryptorchidism was created by extra-abdominal fixation of the gubernaculum in the neonatal period. In group 3, 18 rats underwent a sham operation as controls. At 90 days of age, fertility was then assessed in each rat by housing it with two mature virgin females for 2 weeks. Thereafter, at 115-120 days of age, the rats were killed and their testes removed for histological examination. There was no significant difference in the pregnancy rate between females coupled with rats from any group, but the mean number of offspring was significantly lower in females coupled with rats in group 1 than in group 2. Furthermore, histological examination of both the cryptorchid and contralateral scrotal testes showed more severe changes in the intra-abdominal than the extra-abdominal testes. These results suggest that intra-abdominal cryptorchid testes are significantly more impaired than extra-abdominal cryptorchid testes, and that such impairment might be caused by exposure of the testis to a higher temperature. The more severely impaired undescended testes may thus induce the degeneration of the contralateral scrotal testis and thereby cause subfertility in the intra-abdominal unilateral cryptorchid rat model.

Fertility and histological studies in a unilateral cryptorchid rat model during early and late adulthood.

To investigate the effect of age on fertility and testicular development in rats with untreated unilateral undescended testes. Newborn male Wistar King-A rats were divided into two groups. In group 1, a mechanical model of unilateral undescended testis was created by performing extra-abdominal fixation of the gubernaculum in the neonatal period, and in group 2, sham-operated rats were used as controls. At 90 days old (early adulthood), the fertility of both groups was assessed by mating the rats with mature virgin females for 2 weeks. Thereafter, some of the rats were killed and their testes examined histologically, while the remaining rats were allowed to develop further. At the age of 180 days (late adulthood), fertility was re-assessed in the same way. All the rats were then killed and their testes removed for histological examination. There was no significant difference in pregnancy rate of females mated with males from either group in early adulthood. However, in late adulthood there was a significant reduction in pregnancy rate in females coupled with cryptorchid rats compared with that of females coupled with control rats. There was a significant degeneration of the unilateral cryptorchid testes compared with control testes in both early and late adulthood. In contrast, there were no significant changes in histological development between the contralateral scrotal testes and the controls in early adulthood, although they were significantly different from the controls in late adulthood. These results suggest that fertility is affected by ageing in untreated unilateral cryptorchidism; this may be induced by extensive damage not only in the undescended testes, but also in the contralateral scrotal testis, in this mechanically induced model of unilateral cryptorchidism.

Changes in the Expression Pattern of Luteinizing Hormone Receptor mRNA in Rat Testis during Degeneration of Seminiferous Epithelium

Abstract Previously we reported that the experimental unilateral cryptorchidism altered the expression pattern of LH receptor mRNA in abdominal testis. To clarify whether the change was due to exposure of Leydig cells to high temperature in the abdomen, we examined the effect of unilateral efferent duct ligation on the expression. Two weeks after the operation, a relatively decreased expression of 1.8 kb transcript, increased expression of other transcripts, and degeneration of germ cells were observed in the efferent duct-ligated testes. As these changes were similar to those seen in abdominal testes of cryptorchid rats, exposure of Leydig cells to high temperature was not responsible for the changes in expression pattern of LH receptor mRNA. To examine the correlation between the changes in the receptor mRNA expression and the degeneration of seminiferous epithelium, expression of the mRNA was analyzed in unilaterally cryptorchid rats for 28 days after the operation. In scrotal testes which showed no hi...

Comparison of the effects of vasectomy and experimental cryptorchidism in rats: I. Testicular histology.

The effects of cryptorchidism, one of the most important congenital malformations resulting with the defects in testicular structure thus infertility, is the best known subject in male reproduction. Those best known effects make the usage of cryptorchidism as a second control criteria when the effects of other male infertility reasons are attempted to be determined. Therefore the persistency or reversibility of the effects of vasectomy on testicular structure have been compared to the testicular features following experimental cryptorchidism and to the control group findings. To do this, rats were divided into 3 groups. While the control group animals were sham operated, the experimental group animals were undergone either vasectomy or experimental cryptorchidism procedures. Testicular histology of the control and experimental groups was determined using convenient staining methods at the light microscopical level. The comparison of the findings from the control and experimental groups showed that the testicular features of the vasectomized group were similar to those seen in the control group in contrast to the experimental cryptorchidism group which had irreversible testicular alterations. Results of the present study indicated the convenience of the usage of vasectomy in male contraception.

Degenerative Changes in Spermatogonia Are Associated with Loss of Glucose Transporter (Glut 3) in Abdominal Testis of Surgically Induced Unilateral Cryptorchidism in Rats

Expression of facilitative glucose transporters (Glut 1, 2 and 3) was examined by Western blot analyses 10, 20 and 30 days following surgically induced unilateral abdominal cryptorchidism. The cryptorchid testes exhibited marked degenerative changes in the seminiferous tubules and spermatogonia, impaired and incomplete spermatogenesis and lack of spermatozoa in the lumen. Immunoblotting of testis proteins with Glut transporter antibodies revealed only the presence of Glut 2 and 3 protiens. Glut 2 expression in abdominal testis was increased (45%, 67%, and 40% at 10, 20 and 30 days, respectively) but no significant change was observed in contralateral scrotal testis. Glut 3 expression was reduced by 85-95% compared with contralateral scrotal testis. A significant decrease in Glut 3 levels in abdominal testis was accompanied by an increase in scrotal testis Glut 3 content (80%, 144% and 212% at 10, 20 and 30 days, respectively) compared to age matched control rats. These results suggested that the degenerative changes in abdominal testis may be associated with decreased Glut 3 mediated glucose transport in seminiferous tubules and spermatogonia.

C-89 Studies of Fertility and Histology in Unilateral Cryptorchid Rat Model at Different Ages

C-89 Studies of Fertility and Histology in Unilateral Cryptorchid Rat Model at Different Ages

Prevention of seminiferous tubular atrophy in a naturally cryptorchid rat model by early surgical intervention.

In an attempt to determine whether the seminiferous tubular atrophy of the cryptorchid testis is preventable by early surgical correction of the cryptorchid state, aberrantly developed gubernacula destined to result in a cryptorchid testis in the Long-Evans cryptorchid (LE/ORL) rat were surgically reimplanted to the bottom of the scrotum on day 10 to 12 of age. Testis descent was monitored and the changes in testicular histology and in the volumes of the seminiferous tubules and Leydig cells were examined at day 60. As expected, normal testis descent occurred on or about day 25. Compared to untreated undescended testes at day 60, relative seminiferous tubular volumes (volume: % +/- SEM) were significantly increased by early surgical reimplantation of the gubemacula (89 +/- 1 vs. 66 +/- 3; P < 0.01). Absolute seminiferous tubular volumes (microliter +/- SEM) were also significantly increased by early surgical intervention when compared to undescended nontreated testes (893 +/- 27 vs. 170 +/- 12; P < 0.01). The testes of the surgically corrected cryptorchid animals were similar in all respects to those found in the descended testes of the sham-operated controls. Relative Leydig cell volume (% +/- SEM) was increased in the untreated cryptorchid testes compared to the surgically corrected testes (5.2 +/- 0.6 vs. 1.2 +/- 1.0; P < 0.05). Relative Leydig cell volumes in the surgically corrected testes were not significantly different from those found in the sham-operated descended controls. A modest but significant (P < 0.05) increase in absolute Leydig cell volume was also noted in the cryptorchid testes when compared both to normal controls or surgically corrected cryptorchid testes. From these observations, we conclude that early gubernaculopexy reverses the histologic changes normally seen in the cryptorchid rat testis to a relatively normal histologic architecture. These data provide experimental evidence to support the value of orchiopexy in the treatment of cryptorchidism.

Effects of Unilateral Cryptorchidism on the Expression of Gonadotropin Receptor mRNA

Expression patterns of mRNAs of FSH and LH receptors were examined by Northern blot in adult rat testes after induction of unilateral cryptorchidism. At 4 weeks after the operation, signals of Northern blot were markedly increased in both receptor mRNAs in the abdominal testes as compared to those in the scrotal testes. In addition, there was a remarkable change in size pattern of LH receptor transcripts; the scrotal testes of cryptorchid rats and the intact testes of control rats expressed the transcripts of 7.0, 4.3, 2.5, and 1.8 kb, while the abdominal testes hardly expressed the transcript of 1.8 kb. This finding suggests that the expression of LH receptor mRNA is regulated locally within testis.

The role of the genitofemoral nerve and calcitonin gene-related peptide in congenitally cryptorchid mutant TS rats.

We investigated whether previous gubernacular denervation in TS rats changes the gubernacular contractile response to or binding capacity of calcitonin gene-related peptide receptors. In TS rats there is excessive calcitonin gene-related peptide in the genitofemoral nerve and 85% of the male rats have cryptorchidism. TS rats and controls underwent genitofemoral nerve transection or sham operation at day 0. On day 3 gubernacula were cultured with or without calcitonin gene-related peptide to observe contractions. Gubernacular frozen sections were incubated with 125iodine calcitonin gene-related peptide with or without unlabeled calcitonin gene-related peptide. Exposed x-ray films were quantified by computerized densitometry. In the sham operated controls contractility increased from 15 to 55% by adding calcitonin gene-related peptide compared with 5% and 10%, respectively, in the TS rats. After genitofemoral nerve denervation exogenous calcitonin gene-related peptide increased contractility to 90% in controls and 75% in TS rats. Previous genitofemoral nerve transection increased specific 125iodine calcitonin gene-related peptide binding 21% in controls and about 100% in TS rats. Genitofemoral nerve transection in TS rats restored gubernacular contractile response to calcitonin gene-related peptide in vitro and doubled the binding capacity of calcitonin gene-related peptide receptors.

Multiple factors with steroidogenesis-regulating activity in testicular interbular fluid from normal and experimentally cryptorchid adult rats

Steroidogenesis-stimulating activity (SSA) was examined in testicular intertubular fluid from normal, and short-term and long-term (up to 12 months) experimentally cryptorchid rats, using an in vitro Leydig cell bioassay based on testosterone production over 20 h in the presence of a maximum dose of human chorionic gonadotropin. Total fluid volume increased throughout the period of cryptorchidism, while interbular testosterone concentrations declined. SSA from cryptorchid rats was significantly greater (2- to 3-fold) than normal at all time-points; however, the major increase in activity occurred within the first 4 weeks after treatment. Similar concentrations of lipoproteins were recovered from both untreated and 4-week cryptorchid fluid by density ultracentrifugation, althoug the bioactivity of the cryptorchid testis lipoprotein fraction was 8-fold higher than the lipoprotein fraction from untreated testes. Moreover, removal of the lipoproteins led to a loss of SSA in the lipoprotein-deficient fraction of the intertubular fluid. Consequently, the in vitro bioassay conditions were modified by addition of a constant level of serum lipoproteins to all assay wells. Employing the lipoprotein-supplemented bioassay, multiple stimulatory and inhibitory activities were resolved by Sephadex G-100 gel filtration in intertubular fluid from both normal and cryptorchid testes: (i) an inhibitory activity eluting in the void volume (> 150 kDa), which decreased after cryptorchidism; (ii) a stimulatory activity (40–80 kDa), which did not appear to be affected by cryptorchidism; (iii) an inhibitory activity (20–40 kDa) which decreased after cryptochidism, and (iv) a stimulatory activity (12–20 kDa) which increased after cryptorchidism. These results indicate that the increase in testicular SSA after cryptorchidism is due to an increase in the proportion of stimulatory to inhibitory factors in intertubular fluid, and that both locally-produced and serum-derived factors are involved.

Measurement of immunoglobulin G levels in adult rat testicular interstitial fluid and serum.

Total immunoglobulin G (IgG) in rat testicular interstitial fluid and serum was measured by two-site enzyme-linked immunosorbent assay, which displayed minimal cross-reactivity with IgA or IgM. Normal adult testicular fluid IgG concentrations (2.67 +/- 0.98 mg/ml) were found to be 65% of the levels found in serum. Disruption of seminiferous tubule function by experimental cryptorchidism had no effect on either testicular or serum IgG concentrations. Castration also had no effect on serum IgG concentrations. The effect of the Leydig cell cytotoxin, ethane dimethane sulfonate, on IgG concentrations was obscured by the observation that dimethyl sulfoxide (the carrier solvent) itself effectively eliminated the IgG concentration gradient across the testicular microvasculature. In normal and cryptorchid rats, serum IgG and thymus weights showed no correlation with serum inhibin concentrations measured by radioimmunoassay, although thymus weights were significantly increased in castrate rats. The data confirm that the permeability of the rat testis to circulating IgG is relatively high compared with other tissues and suggest that the testicular transendothelial transfer of IgG is unaffected by changes in testicular activity or hormone secretion. The high intratesticular concentration of IgG and its apparent lack of local regulation provide further evidence that the phenomenon of immune privilege in this tissue cannot be attributed simply to restriction of immune effectors from the testicular environment. The data also indicate that serum IgG levels in the adult male rat are not influenced in the short-term by changes in peripheral testicular androgen levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimentally Induced Cryptorchidism Increases Apoptosis in Rat Testis1

Although surgical induction of cryptorchidism in the rat is known to cause infertility due to disruption of spermatogenesis, the exact cellular mechanism responsible for the degenerative changes in cryptorchid testes is unclear. Using a sensitive autoradiographic method for the detection of apoptotic DNA fragmentation, we have investigated the effect of experimentally induced cryptorchidism on apoptotic cell death in testes of immature rats. Bilateral or unilateral cryptorchidism decreased the weight of affected testes within 4 days; these decreases (24-27%) became significant (P < 0.05) at 7 days after the operation. Testes of sham-operated animals contained predominantly high molecular weight DNA (> 15 kb), whereas DNA cleavage into low molecular weight ladders characteristic of apoptosis was increased by induction of bilateral cryptorchidism in a time-dependent manner, i.e., 2.0-, 2.8-, and 4.2-fold (p < 0.05) at 2, 4, and 7 days after operation, respectively. In unilaterally cryptorchid animals, sham-operated testes also contained predominantly high molecular weight DNA, whereas induction of cryptorchidism of the contralateral testes increased DNA cleavage into low molecular weight fragments 3.0-, 2.8-, and 3.9-fold (p < 0.05) at 2, 4, and 7 days after the operation, respectively. In situ analysis of DNA fragmentation in testes of unilaterally cryptorchid rats at 7 days after the operation indicated that germ cells, mainly primary spermatocytes were affected and that the percentage of seminiferous tubules containing labeled cells increased in the operated testis as compared to the contralateral control in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of normal sexual dimorphism of the genitofemoral nerve spinal nucleus in the mutant cryptorchid (TS) rat.

Sexual dimorphism of the genitofemoral nerve spinal nucleus has been demonstrated in normal rodents. Calcitonin gene-related peptide is a neurotransmitter, present in the genitofemoral nerve, that has been implicated in the regulation of gubernacular migration and inguinoscrotal testicular descent. A combination of retrograde fluorescent labelling of the genitofemoral nerve and immunohistochemistry for calcitonin gene-related peptide was used in 1-3-day-old mutant TS rats with 85% incidence of congenital cryptorchidism and an absence of the normal sexual dimorphism of the genitofemoral nerve spinal nucleus was demonstrated. There was no significant difference between male and female nuclei with respect to fluorescent-labelled neurones as well as those immunoreactive for calcitonin gene-related peptide, in contrast to an obvious sexual dimorphism present in normal control animals. This lack of normal sexual dimorphism of the genitofemoral nerve nucleus is likely to be important in the pathogenesis of cryptorchidism in this animal model.

Capsaicin Increases the Frequency of Cryptorchidism in Flutamide-Treated Rats

Capsaicin, a specific neurotoxin that irreversibly results in degeneration of sensory C fibers of the peripheral nerves, was investigated for its ability to affect testicular descent. In particular, we investigated the ability of prenatal flutamide, postnatal capsaicin or combined prenatal flutamide with postnatal capsaicin to induce undescended testes in rats. Within these treatment groups the frequency of maldescent was 42% in the animals treated prenatally with flutamide, 32% with postnatal capsaicin, and 100% with prenatal flutamide and postnatal capsaicin. Of interest, it was found that all animals that received capsaicin had maldevelopment of the scrotum. Our data suggest that capsaicin increased the frequency of cryptorchidism in rats prenatally treated with flutamide, perhaps by inhibition of scrotal development. These findings indicate that sensory nerves to the scrotum may affect scrotal development and testicular descent.

Division of the genitofemoral nerve in unilateral cryptorchid rats

The long-term effect of dividing the genitofemoral nerve supply to the undescended testis in naturally cryptorchid rats is reported. 20 male Buffalo rats with an undescended right testis, noted at the age of 30 days, were used for the investigation. 20 healthy animals served as controls. The abdominal division of the right genitofemoral nerve was performed in 10 cryptorchid and 10 healthy animals at the age of 40 days. The animals were mated at 150 days of age. Both testes were removed at 180 days of age. In each excised testis the testicular weight and seminiferous tubular diameters were measured, and the maturity of the germinal epithelium was determined using the Johnsen testicular biopsy score. The experiment showed that in 70% of the nonoperated cryptorchid rats, both testes were affected at the age of 180 days, and 90% of these animals were infertile. In the operated cryptorchid rats, the contralateral descended were not damaged, and 60% of these animals were fertile. The division of the genitofemoral nerve in healthy animals had no effect. The results suggest that neural transmissions might play a role in the damage to the descended testis in unilateral cryptorchidism. This observation requires further investigations.

The effect of experimental cryptorchidism on the phosphorus NMR spectrum of the rat testis.

Magnetic resonance (MR) spectroscopy of the cryptorchid rat testis was used to test whether changes in the MR spectra of the rat testis might be a more sensitive indicator of changes in the metabolic status of germ cells in the testis rather than simply the cell types present. Testes of adult Wistar rats before and during 42 days of experimental cryptorchidism were investigated by in-vivo 31P MR spectroscopy. Results were compared to MR studies of the synchronized developing testis. The testicular phosphomonoester/ATP (PM/ATP) ratio was dependent only on the cell types present, and showed the same characteristics for each cell type present in the degenerating testis as in the developing testis. The testicular phosphodiester/ATP (PD/ATP) ratio decreased rapidly when the number of round and elongated spermatids was reduced. Similar effects, although less pronounced, were seen in the developing testis. The pH decreased rapidly after cryptorchidism, and was related inversely to the PM/ATP ratio, which was also observed in the developing testis. This study demonstrates that MR spectroscopy monitors the cell types present in the rat testis rather than its metabolic status.

Comparison of the ultrastructure of the cremasteric muscle‐scrotal interface in normal and cryptorchid rats following division of the neonatal genitofemoral nerve

AbstractThe gubernaculum, innervated by the genital branch of the genitofemoral nerve, plays an important role in inguinoscrotal testicular descent. Cremaster muscle develops within the gubernaculum, and it has been demonstrated that an intact nerve is essential for gubernacular migration and scrotal pouch formation. In this electron microscopy study, we investigated the relationship between the gubernaculum, cremaster muscle, and the developing scrotum in normal rats and rats rendered cryptorchid by genitofemoral nerve division.In 12 neonatal rats, the genitofemural nerve was divided unilaterally. Rats were sacrificed at 2, 4, 7, 10, 14, and 18 days (n = 2 for each group), and sagittal sections through the cremaster muscle‐scrotal interface evaluated with transmission electron microscopy. These were compared to non‐operated control animals sacrificed at 0, 2, 4, 7, 10, 14, and 18 days (n = 2 for each group). We observed the following changes in the cremaster muscle of the operated group compared to unoperated controls: (1) an increase in the number of immature muscle fibers; (2) variable muscle fiber size; (3) disorganization of internal structures; and (4) vacuolization within the muscle fibers. There were no demonstrable ultrastructural interface or scrotal ligament differences between the operated and unoperated specimens.This study suggests that architecturally normal early gubernacular and cremaster muscle growth is dependent on an intact genitofemoral nerve. The poor scrotal growth seen without genitofemoral nerve input may be due to attachment of the scrotal ligament in an ectopic location in combination with abnormal cremaster muscle development. © 1994 Wiley‐Liss, Inc.