Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide.

Active immunization to immunomodulate regulatory processes suffers from the disadvantage that the antigen is usually 'self' and therefore poorly immunogenic. This has been well illustrated by the long-standing experience with immunocastration vaccines targeting GnRH, a ten amino acid peptide. Not all animals vaccinated with these vaccines are equally affected, even after multiple vaccinations. This is a severe handicap when immunocastration vaccines are applied to male piglets to circumvent surgical castration. Surgical castration is universally practised to prevent boar taint, produced in the testicles of mature boars. Alternative immunocastration is only acceptable if all animals are equally affected using a minimum of vaccinations. Vaccines based on the GnRH peptide itself cannot meet these goals. We showed that using a GnRH-like peptide, a 20 amino acid tandem repeat of the amino acid sequence of the GnRH peptide, these goals can be attained. Using the tandem GnRH peptide to vaccinate male piglets completely abolished the development and endocrinological functioning of the testicles, in contrast to monomer GnRH. These results show that superior antigens can be made for effective immunomodulation by appropriate alteration of the antigen.

Lesions in the hypothalamus after active immunisation against GnRH in the pig.

The terminals of the hypothalamic gonadotrophin hormone-releasing hormone (GnRH) neurons are located within the median eminence and thereby extend beyond the protection of the blood-brain barrier. Thus, these terminals may be subjected to direct autoimmune action in animals that are actively immunised against GnRH. Boars (male pigs) (n = 108) were actively immunised against GnRH by two successive injections with synthetic GnRH, covalently coupled to KLH and dissolved in CFA or IFA. They were killed at 26 weeks of age. Immunised boars were selected on the basis of the resultant testes size, which indicates the effectiveness of the immunisation. The hypothalami of 25 selected animals were studied by histological and immunocytochemical techniques and compared with the hypothalami of three sham- and nine control animals. In the immunised animals, changes in the GnRH system had taken place. These comprised dystrophy of the perikarya and a sharp decrease of the GnRH immunocytochemical reactivity in the terminals within the median eminence. In addition, various degrees of inflammatory reactions were present, particularly within the median eminence. These consisted of tissue disruption by edema, collapse of the capillaries, fibrosis and infiltration with fibroblasts. In addition, accumulations of neurosecretum within the median eminence in combination with hypertrophy of magnocellular neurons within the hypothalamus were present. The reactions were restricted to the median eminence and did not involve other neurohemal organs or other parts of the hypothalamus. A correlation could be established between the incidence of the lesions and the effectiveness of the GnRH autoimmunity (as indicated by the size and endocrine function of the gonads and the anti-GnRH titres). Changes in extra- and intracellular IgG immunocytochemical reactivity within the median eminence indicated the involvement of IgG. The effects were absent from control and sham vaccinated animals and after vaccinations with other compositions of the vaccine. Thus, hypothalamic lesions have been observed in this selected group of animals, vaccinated against GnRH with this particular vaccine.

Repeated interruptions of the testicular blood flow do not have long-term effects on spermatogenesis in the ram.

The effect of repeated interruptions of the testicular blood flow on spermatogenesis was studied in mature Texel rams. Reversible interruption of the blood flow was achieved by an inflatable occluder, placed around the testicular artery at the level of the spermatic cord. In eight testes the blood flow was successfully interrupted six times for 1 h within 3 weeks and in 14 testes nine times for 1 h within 3 weeks. Nine weeks after the last blood flow interruption spermatogenesis was evaluated in histological sections of the testes. Both after six and nine blood flow interruptions a qualitatively complete epithelium was found in at least 90% of the seminiferous tubules. Cell counts in stages VII and VIII of the spermatogenic cycle revealed a slight decrease of spermatocytes and spermatids in the tubules with a complete epithelium after nine occlusions, which was only statistically significant for Preleptotene Spermatocytes. After six occlusions the numbers of all cell types were at or even slightly above control levels. These results show that repeated periods of ischaemia for 1 h do not result in conspicuous long-term damage to spermatogenesis.

Protection of spermatogenesis against cytotoxic effects of two chemotherapeutic drugs by temporary testicular blood flow interruption in the ram.

Temporary interruption of the testicular blood flow for 1 h after injection of cytostatic drugs has a protective effect on spermatogenesis. This was shown in experiments in which spermatogenesis was evaluated at four weeks after a single intravenous injection of Adriblastina (ADR; doxorubicin hydrochloride) or Mitomycin-C-kyowa (MIT). Interruption of the blood flow was performed by inflation of an occluder implanted around the testicular artery. The animals were killed and histological sections prepared 4 weeks after treatment. In all drug-treated animals spermatids were near absence and spermatocytes were decreased in number. Therefore, even after occlusion of the blood flow, the drug doses were high enough to kill not only large numbers of differentiating spermatogonia but also stem cells. The response of the stem cells to the treatments was evaluated by counting the numbers of A spermatogonia per 100 Sertoli cells in the different groups. Normal numbers of these cells were found after both MIT and ADR, indicating that the stem cell population had responded to the initial cell loss by extra proliferation. However, significantly higher numbers of A spermatogonia were found in the drug-treated animals in which the testicular blood flow was interrupted for 1 h. This indicates that occlusion of the blood flow to the testis for 1 h results in a faster recovery of spermatogenesis than after drug treatment alone.

Effects of varicocele treatment in adolescents: a randomized study.

OBJECTIVE: To study the effects of varicocele treatment on testicular function in adolescents. DESIGN: A prospective controlled study in 88 randomly selected adolescents. SETTING: All participants were referred to the fertility outpatient clinic of our university hospital. PARTICIPANTS: All participants with a varicocele were randomly assigned into two groups. Group 1 (n = 33) was not treated, whereas group 2 (n = 34) was treated. A similar group of healthy volunteers without a varicocele served as a control group (group 3, n = 21). INTERVENTIONS: Testes volumes were measured at intake and during follow-up using an orchiometer. Semen analysis was performed according to standard procedures both at intake and after 1 year of follow-up. Serum hormone levels were determined at intake using a radioimmunoassay. Treatment was performed by means of transcatheter embolization of the left testicular vein. MAIN OUTCOME MEASURES: Testes volumes and semen quality at intake and after 1 year of follow-up were compared within and between the three groups. Hormonal parameters were determined at intake only. RESULTS: Before treatment, the mean left testis volume in groups 1 (n = 26) and 2 (n = 27) (20.0 mL; 95% confidence interval [CI]: 18.2 to 21.8 and 21.6 mL; 95% CI: 19.4 to 23.8, respectively) were significantly smaller than those in the control group (n = 19) (24.5 mL; 95% CI: 22.7 to 26.4). During follow-up, left testis volumes of the treated group were comparable with those in the control group (24.2 mL; 95% CI: 22.2 to 26.1 and 24.8 mL; 95% CI: 23.0 to 26.7 respectively) and significantly (P < 0.001) different from the untreated group (20.3 mL; 95% CI: 18.8 to 21.8). A significant increase in left (P < 0.01) as well as right (P < 0.05) testis volume was observed after treatment. Semen parameters before treatment were not significantly different between the three groups. Sperm concentration increased significantly (P < 0.01) from 47.4 x 10(6)/mL (95% CI: 42.5 to 53.3) to 68.9 x 10(6)/mL (95% CI: 50.6 to 87.2) in the treated group, whereas semen quality in the untreated and control groups did not change. Although both testes volumes and sperm concentration improved in the treated group, these phenomena were not consistently correlated to each other. CONCLUSIONS: Although not apparent in all adolescents, varicocele correction resulted in an increase in left testis volume and sperm concentration. At this moment, it is not clear if early preventive treatment of varicocele in adolescents, in time, will have a positive effect on testicular function.

Hormone-induced resistance of rat Leydig cells to the cytotoxic effects of ethane-1,2-dimethane sulphonate.

Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 +/- 40 to 1368 +/- 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 +/- 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.

Pubertal development in the male pig: effects of treatment with a long-acting gonadotropin-releasing hormone agonist on plasma luteinizing hormone, follicle stimulating hormone and testosterone.

The effects of a long-acting gonadotropin-releasing hormone (GnRH) agonist, [D-Trp6]-GnRH (GnRH-A) on developmental profiles of plasma luteinizing hormone (LH), follicle stimulation hormone (FSH) and testosterone (T), and pituitary responsiveness to exogenous GnRH were studied in male Dutch Landrace x Large White crossbred pigs from 1 to 30 wk of age. Group 1 control animals (control; n = 12) were injected subcutaneously in the neck with vehicle at 1 and 16 wk of age. Group 2 animals (early treatment; n = 10) were injected with 600 micrograms [D-Trp6]-GnRH at 1 wk and with vehicle at 16 wk. Group 3 animals (late treatment; n = 8) were injected with vehicle and 3 mg GnRH-A at 1 and 16 wk, respectively. Group 4 animals (early plus late treatment; n = 9) were injected at both 1 and 16 wk with GnRH-A. Blood was collected by brachiocephalic puncture at weekly or biweekly intervals, and through brachiocephalic cannulae, to determine longitudinal profiles of LH, FSH and T, and plasma gonadotropin responses to intravenous injection of GnRH (0.1 microgram/kg), respectively. In control animals, LH and FSH declined over the first 5 wk of postnatal life and peaked again at 10-14 wk. Levels of both hormones were basal from 18 to 30 wk. Plasma T was high in the first week, declined progressively over the next few weeks and remained low until 24 wk when a transient increment was noted. The LH and FSH responses to acute GnRH stimulation were similar at 7 and 14 wk and declined significantly at 23 wk of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Testis volumes, semen quality, and hormonal patterns in adolescents with and without a varicocele.

OBJECTIVE: To study the effects of varicocele on testicular function in adolescents. DESIGN: A prospective controlled study in 88 randomly selected adolescents. SETTING: All participants were referred to the fertility outpatient clinic of our University Hospital. PARTICIPANTS: All participants with a grade II varicocele (group 2) or a grade III varicocele (group 3) were selected at a district military medical council, whereas a similar group of healthy volunteers without a varicocele served as controls (group 3). INTERVENTIONS: Testis volumes were measured using an orchiometer. Semen analysis was performed according to standard procedures, and serum hormone levels were determined using a radioimmunoassay. MAIN OUTCOME MEASURE(S): Testis volumes, semen quality, and hormonal parameters in adolescents with and without a varicocele were compared. RESULTS: In group 1 (n = 21), the mean left testis volume (24.5 mL; 95% confidence interval [CI]: 22.8 to 26.2) was significantly (P less than 0.05) different from group 2 (n = 15) (20.9 mL: 95% CI: 18.5 to 23.4) and group 3 (n = 52) (20.7 mL; 95% CI: 19.2 to 22.2) (P less than 0.01) adolescents. In adolescents with a pronounced varicocele-associated left testicular growth failure, the total sperm number was reduced. However, sperm concentration, motility, and morphology were not altered. Luteinizing hormone, follicle-stimulating hormone, testosterone, and prolactin levels were all within the normal ranges in the three groups. CONCLUSIONS: Left testicular growth failure in adolescents with a varicocele is only associated with a decrease in total sperm number.

Total ligation of the left renal vein in the dog: an inappropriate model for varicocele.

Induction of varicocele was attempted by ligation of the left renal vein (LRV) in male dogs (Group I). Before the operation and in the 4-month post-operative period, sperm count, sperm motility, and sperm morphology of Group I (n = 8) dogs were compared to sham operated animals (Group II, n = 5). Furthermore, haemodynamics as well as testicular and vascular morphology were studied. In Group I, changes in diameter and consistency of the spermatic cord were temporary. Semen quality was reduced significantly during the second month after ligation of the LRV, but improved thereafter. Haemodynamic studies revealed that LRV blood pressure was increased significantly in Group I dogs. An extensive venous collateral network replaced the occluded LRV. Retrograde blood flow in the left testicular vein (LTV) was observed only in the proximal part of the LTV of Group I dogs. In Group II dogs numerous pairs of sufficient valves prevented reflux into the LTV. Histological examination revealed that spermatogenesis was not impaired and that the left pampiniform plexus had not changed. The number of Leydig cells was decreased slightly in Group I dogs. Sufficient valves in the LTV prevented formation of a permanent varicocele.

Development of a new Leydig cell population after the destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study.

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3 or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered. In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Triploidy and intersexuality in adult commercial layers.

The cause of masculinisation of the left ovary and the outgrowth of the vestigial right gonad was investigated in intersexual hens. Tumour-like cell masses, resembling mouse tubular adenomas of the ovary, were observed in the majority of masculinised left gonads. Except for one male and two intersexuals, testosterone concentrations were below detectable levels. For oestrogen, progesterone and the oestrogen : progesterone ratio all differences were significant, except for the difference between the intersex and the male. Histochemically this cell mass showed weak androgen-synthesising activity. These intersexual gonads showed similarities to normal testicular tissue. Karyotyping revealed 3n-autosomes and a ZZW sex chromosome constitution. The early and almost complete absence of cortical follicular structures was most notable and may have been the cause of the sex reversal.

Stimulation of the proliferation and differentiation of Leydig cell precursors after the destruction of existing Leydig cells with ethane dimethyl sulphonate (EDS) can take place in the absence of LH.

In hypophysectomized rats, 2 days after the administration of the cytotoxic drug ethane dimethyl sulphonate (EDS), the proliferative activity of Leydig cell precursors increased six-fold. Thus, factors other than LH act locally to stimulate the proliferation of precursor cells after EDS. Twenty-six days after EDS administration, neither cells with the morphological characteristics of Leydig cells nor histochemical enzyme activities, such as 3 beta-HSD and alpha-naphtyl esterase, could be detected in testis tissue. In hypophysectomized rats treated daily with hCG (100 iu) for 7 days, starting at 26 days after EDS, the number of Leydig cells was increased to 48 +/- 11 cells (per 1000 Sertoli cells), which is approximately 4.5% of the intact control level. 3 beta-HSD and alpha-naphtyl esterase activity could be detected, and plasma testosterone levels had increased 15-fold compared with the hypophysectomized controls. These results show that proliferation and some differentiation of precursor cells along the Leydig cell lineage can occur independent of LH, but the final stages of the differentiation process require hCG stimulation.

Growth and differentiation of the gubernaculum testis during testicular descent in the pig: changes in the extracellular matrix, DNA content, and hyaluronidase, beta-glucuronidase, and beta-N-acetylglucosaminidase activities.

The gubernaculum testis is a loose connective tissue organ that plays an essential mechanical role in testicular descent. In the pig, the first phase of descent (transabdominal migration) is brought about by growth of the gubernaculum through the inguinal canal into the scrotum and simultaneous somatic growth of the fetus. During the second phase the gubernaculum condenses, thus allowing the testis to descend into the scrotum. The nature of gubernaculum development (growth and differentiation) was investigated with respect to cell proliferation, extracellular matrix (ECM) composition, and acid hydrolases. Deoxyribonucleic acid (DNA) was used as a measure of cell number and hydroxyproline (HYP) was an estimate of interstitial collagen. The first phase of gubernaculum development was characterized by rapid cell proliferation and concomitant synthesis of sulphated glycosaminoglycans (S-GAG), hyaluronic acid (HA) and collagen. During the second phase cell proliferation ceased and DNA concentration increased. The amount of S-GAG remained closely related to the amount of DNA while HYP increased further. However, HA decreased during the second phase and thus HA metabolism seems to play a crucial role in biphasic development of the gubernaculum. The activities of the enzymes that are needed for biodegradation of HA (hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase) were measured in gubernaculum homogenate from animals during the first and second phase of testicular descent. These enzymes were detectable in gubernaculum and rose during the second phase of testicular descent. It was concluded that a very distinct dichotomy in the nature of gubernaculum development during the first and second phase could be discerned with respect to cell proliferation rate and ECM synthesis and degradation. These observations provide useful tools for future in vivo and in vitro investigations into the process and regulation of testicular descent.

Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: the role of LH/human chorionic gonadotrophin.

The influence of LH levels on the proliferation and differentiation of possible Leydig cell precursors was investigated in adult rats, after the destruction of the existing Leydig cells with the cytotoxic drug ethane dimethyl sulphonate (EDS). In rats bearing a testosterone implant which prevented the rise in plasma LH levels and kept them within the normal range after the destruction of the Leydig cells, the proliferative activity of possible Leydig cell precursors still increased seven- to eightfold 2 days after EDS administration. Apparently, in this situation, locally produced factors, and not LH, may play a role in the stimulation of proliferation. The proliferative activity of the possible precursor cells could be further stimulated by treating rats with daily injections of human chorionic gonadotrophin (hCG) following EDS administration. It was concluded that the proliferative activity of possible Leydig cell precursors is probably regulated by both paracrine and endocrine factors. Almost no Leydig cells were formed in the rats bearing a testosterone implant during the first 4 weeks after EDS administration. When these rats were treated with hCG, starting 28 days after administration of EDS, a substantial number of Leydig cells was found after 2 days, and these cells also showed 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and alpha-naphtyl esterase (alpha-NE) activity. When hCG treatment was started at 14 or 21 days after EDS administration, some cells with the nuclear characteristics of Leydig cells were present after 2 days, but no 3 beta-HSD or alpha-NE activity could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of direct effects of GnRH on testicular steroid secretion in the ram.

Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0.5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.

Induction of varicocele in the dog: I. Partial ligation of the left renal vein does not induce a varicocele in the dog.

Induction of varicocele was attempted by partial ligation of the left renal vein in 10 male dogs. The effects on sperm count, sperm motility, and sperm morphology, as well as on hemodynamics, were assessed. Furthermore, testicular, vascular, and kidney morphology was studied. Changes in the diameter and consistency of the left spermatic cord were found to be temporary. Total sperm count, sperm motility, and the total number of oval forms were not significantly altered. Hemodynamic studies revealed a renocaval pressure gradient, but retrograde flow in the distal part of the left testicular vein could not be observed by arteriography. A collateral network was found to compensate for the restricted left renal vein. Histologic examination revealed no damage to the seminiferous epithelium. Changes were not found in the kidney and left pampiniform plexus. Although some temporary changes induced by the partial ligation of the left renal vein are suggestive of varicocele, this hemodynamic study shows that the presented dog model does not mimic varicocele as encountered in man.

Turnover time of Leydig cells and other interstitial cells in testes of adult rats.

The aim of this study was to investigate the turnover of Leydig cells and other interstitial cells in the adult rat testis. Normal adult rats received injections of [3H]thymidine at 9:00 and 21:00 for 2, 5, or 8 days. The percentage of labeled Leydig cells, which was initially low (0.8% +/- 0.2%), gradually increased during treatment to 1.4% +/- 0.3%. The percentage of labeled peritubular cells was considerably higher and increased from 1.4% +/- 0.1% to 3.5% +/- 0.4% during [3H]thymidine treatment. The remaining interstitial cells were the most actively proliferating cells: the percentage of labeled cells increased from 2.4% +/- 0.2% to 7.5% +/- 0.8% during [3H]thymidine treatment. Leydig cells, peritubular cells, and the remaining interstitial cells in the adult rat testis undergo proliferation. By means of a linear regression analysis and an arcsin transformation, an estimation of the time interval needed to replace various types of interstitial cells was obtained. Taking into account the 95% confidence interval, the turnover time of Leydig cells ranged from 142 to 2823 days. The calculated turnover time for the peritubular cells was 85 to 257 days.

Effects of pure FSH and LH preparations on the number and function of Leydig cells in immature hypophysectomized rats.

The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17-18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 micrograms FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 micrograms) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3 beta,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3 beta-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.(ABSTRACT TRUNCATED AT 250 WORDS)