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Age-related changes in reproductive hormones and in Leydig cell responsivity in the male Fischer 344 rat.

Age-related changes in testicular responsiveness to pituitary hormone stimulation were measured in Fischer rats. Suspensions of dispersed Leydig cells were prepared from the testes of groups which were 5, 12 and 29 months old. The suspended cells were challenged with 250, 750 and 2000ng Monkey-LH (LER 1909-2) for two hours and the medium subsequently assayed for testosterone. Concentrations of the hormone were determined per million cells in suspension. Besides measurement of testosterone production by each age group, pituitary and plasma LH, FSH and prolactin, and plasma testosterone were also assayed by radioimmuno-technique. Total testis weight increased in the oldest animals as the result of a high incidence of gonadal pathology. If tumorous tissue was excluded, however, testis weight fell for this group. Also, significant decreases in Leydig cell number were apparent in 12 and 29-month-old rats. Basal testosterone production, on a per million cell basis, however, was not significantly differnet in young and middle-aged rats, nor was the change in the amount of steroid produced by LH stimulation of the isolated Leydig cells. Seminal vesicle weight, prostate weight and serum testosterone decreased with age. Similarly, pituitary LH and FSH concentration decreased, but pituitary prolactin increased. Histological changes and pituitary hypertrophy reflected this increase in prolactin concentration. It is proposed that decreased testicular function in old age may not totally be the result of a decrement in Leydig cell steroid producing capacity, but that it may also occur due to Leydig cell loss, possibly as the result of age associated changes in the brain and pituitary.

Aging↗

Biphasic effect of testosterone propionate on Sertoli cell secretory function.

When various doses of testosterone propionate (10 to 10,000 mug/day) were given to 21-day-old rats for 10 days a biphasic effect was seen both on testis weight and production of androgen-binding protein (ABP). At low doses (10 to 100 mug testosterone propionate/day) there was a reduction in testis weight as well as ABP content in the epididymis. At higher doses of testosterone propionate, there was a stimulation of both testicular weight and ABP production in spite of suppressed serum FSH and LH levels. These effects of testosterone propionate on Sertoli cell secretory function strongly suggest that the Sertoli cell is a target cell for androgen.

Animals↗

Urinary creatine as a possible marker for testicular damage: studies with the testicular toxic compound 2-methoxyethanol.

A single dose of 500 mg/kg of 2-methoxyethanol given to male rats caused testicular damage, detected by histopathology, a significant reduction in relative testis weight, and a significant rise in urinary creatine, maximal at 48 h after dosing. Further studies showed that the reduction in relative testis weight and creatine excretion were dose related. Conversely, urinary creatinine showed a dose-related decrease at 24 h after dosing. In female rats dosed with 500 mg/kg of 2-methoxyethanol, there was no increase in urinary creatine or decrease in creatinine. These data suggest that urinary creatine may be a useful, noninvasive marker of testicular damage.

Animals↗

Effects of low intensity red light on testicular recrudescence in Japanese quail.

Experiments were conducted to determine if sighted and blind plumage color mutants of Japanese quail elicit comparable testicular recrudescence when exposed to long daily photoperiods of low intensity red light. Quail exhibiting testicular regression after exposure to 8 hr of light daily for several weeks were blinded via bilateral ocular enucleation. A control group was not enucleated. Two light intensities, 1.4 and 2.8 lx, were applied to half of the birds in each group during a 16 hr daily photoperiod for several weeks. Enucleated birds and birds under the 2.8 lx intensity had greater testis weights than the controls. Birds were also blinded by optic tract sectioning, and a .7 lx red light intensity treatment was included. Greater testis weights were found in birds under higher light intensities. Three plumage pigmentation mutants, sex linked incomplete albino, white and wild-type, were exposed to 8 hr of light daily for either 4 or 8 weeks to induce testicular regression. They were then exposed to the three light intensities for 3 weeks. Higher intensities and albinism resulted in greater testicular recrudescence only when the regression interval was 8 weeks, even though testicular regression appeared after 4 weeks. The absence of eyes or plumage pigmentation, resulting in an increased exposure of the encephalon for extraretinal light reception, may have effected greater testicular recrudescence. Severed optic tracts with tract eyes did not produce this response to light, indicating that the eyes may play a role in mediating the photosexual reflex in quail. Lower intensities may have resulted in reduced tissue penetration and testicular growth. A definite threshold intensity necessary to elicit testis growth was not found.

Animals↗

Position and histological structure of the testes in the brown hare (Lepus europaeus) during seasonal regression and recrudescence.

The position and histological structure of the testes of 33 brown hares (Lepus europaeus) were studied from July to December. From July to September, the testes were located in the scrotum; in October and November, in some animals, the testes were positioned more or less in the inguinal canal towards the abdominal cavity, and in December none of the investigated animals had testes located in the scrotum. Testes were weighed and a quantitative analysis of tissue components was performed: the diameter of the seminiferous tubules, the depth of the seminiferous epithelium, the thickness of the tunica albuginea, the thickness of the peritubular tissue and the relative proportion of seminiferous tubules were determined. The tunica albuginea and peritubular tissue were thickest in September, October and at the beginning of November. In the same months the testis weight was low, and the diameter of the seminiferous tubules, the depth of the seminiferous epithelium and the relative proportion of seminiferous tubules in the testis tissue were significantly lower than in other months. We did not find any correlation between testicular regression or testis weight reduction and the change in the position of the testes. During recrudescence of spermatogenesis in November and December the testes were located in the inguinal canal.

Animals↗

Failure of cryptorchidism to suppress fertility in a tropical rodent.

The cane mouse (Zygodontomys brevicauda) breeds year-round on the hot llanos of Venezuela, only 8 degrees above the equator. The reproductive responses of the males of this species to heat were compared with those of a temperature zone rodent, the white-footed mouse (Peromyscus leucopus). When tested at different ambient temperatures, the movement of the testis in relation to the scrotum was similar in the two species, but the cane mouse's testis proved to be much more resistant to maintenance at core body temperature. In two experiments, cryptorchid cane mice experienced only a 24% and a 5% decrease in testis weight, and almost all of these males showed normal spermatogenesis and sperm storage. In comparison, cryptorchid white-footed mice showed a 58% drop in testis weight, a total inhibition of spermatogenesis, and a complete or near absence of stored sperm in all males. A mating test demonstrated that cane mice indeed remain fertile for at least 2 to 3 mo after being rendered cryptorchid. Normal numbers of these males when paired with females fathered normal litters. It is noted that the relative insensitivity of the testis of the cane mouse to heat might actually be more representative of scrotal mammals in general than is suggested by our present perspective, which has been developed on the basis of study of humans and mammals from cool climates rather than the tropics, where most mammals live.

Animals↗

[Stress caused by immobilization and low protein intake in rats. Morphological changes].

The periodic immobilization of the animals receiving a control diet resulted in a smaller weight gain than in controls. The animals fed a protein deficient diet gained also less weight as compared to controls. The association of the deficiency and the stress of immobilization further decreases the weight gain, but the differences were not significant. The animals submitted to restraint or protein deficiency stress showed an increase in their heart weight in relation to body weight. This increase in more significant when immobilization and protein deficiency were associated. The degree of cardiac muscle fibers damage was also found more pronounced in this group of animals, when analysed through histochemical methods. Liver weight was not found different in the animals submitted to the experimental program. Histological examination of this organ showed fatty infiltration in the protein deficient animals. The picture was not found to be agravated when the animals were further stimulated by the restraint mechanism. Spleen weight was found to be similar in all groups. Histological study of the protein deficient group showed small lesions, that were increased when the deficiency was associated to immobilization. The relative testis weight of the restraint animals receiving the control diet was found to be similar to the animals not immobilized. The protein deficient animals have significantly smaller testis weight than the well nourished group. The histologic study showed that restraint or protein deficiency are responsible for a sexually retarded animal, but the lesions increased with the association of restraint and deficiency.

Animals↗

Postnatal hypothyroidism does not affect prepubertal testis development in boars.

Young boars were treated with propiothiouracil to induce hypothyroidism to examine its effects on postnatal testicular development. Treatments with 0.1% 4-propyl-2-thiouracil (PTU) in drinking water started after weaning, at 3 weeks of age and all boars were severely hypothyroid at 6 weeks of age as determined by measuring T3 and T4 in blood plasma. Boars were castrated at different ages up to 20 weeks and their testes used for histological and immunohistochemical analyses. Although small but significant reduction in testis weight was observed from 8 to 12 weeks of age, this was not accompanied by significant difference in testicular volume. By 20 weeks of age, at the beginning of puberty, the differences in testis weights between control and treated groups of boars disappeared suggesting there is no lasting effect of hypothyroidism on postnatal development of boar testis. Immunohistochemical staining was used to determine the presence of molecular markers in both Sertoli and Leydig cells. Again, there were no differences between testes from control and treated boars in the pattern or intensity of immunostaining using antibodies against 3beta-hydroxysteroid dehydrogenase, antimullerian hormone or proliferating cell nuclear antigen (PCNA). Immunostaining with antibodies against PCNA showed interesting results as it was observed that Sertoli cells still express this marker of proliferating cells at 14 weeks of age, later than previously suggested cessation of Sertoli cell proliferation. This study suggests that hypothyroidism in boars does not have similar effects on postnatal testis development as reported in some other species.

Animals↗

Effect of melatonin administered during the night on reproductive function in the Syrian hamster.

Syrian hamsters were maintained on a long photoperiod (lights on 0600-2000 h) and injected daily with melatonin (10 microgram per injection) for 6-7 weeks. Females injected at 2000 h or 0400 h ceased estrous cyclicity, and males treated similarly at these times had reduced testis weights by the sixth week of treatment. Daily administration of the same dosage of melatonin at 2400 and 0100 h failed to interrupt estrous cyclicity or reduce testis weight. These observations suggest that exogenous melatonin administered at 2000 h or 0400 h may be temporally adding to endogenous circulating melatonin, inducing gonadal quiescence.

Animals↗

Effect of melatonin infusion duration and frequency on gonad, lipid, and body mass in pinealectomized male Siberian hamsters.

The goal of this study was to discriminate between two hypotheses regarding how the circadian rhythm of pineal melatonin (MEL) production transmits photoperiodic information: (1) A circadian rhythm of sensitivity to MEL regulates the hormone's effect; (2) the duration of the MEL signal, rather than its circadian timing, is the critical parameter of the MEL rhythm. The experiment examined the response of pinealectomized (PINX) male Siberian hamsters to 10-hr (short-day-type) versus 6-hr (long-day-type) duration MEL infusions (10 ng/infusion) in cycles with period lengths (T) of 18, 24, 36, and 48 hr. After cannula implantation, animals were moved from LD 16:8 to LD 10:14 (lights-on from 0500 to 1500 hr, EST), where the timed infusions began. Additional T 24 cycles included as controls employed 18-hr MEL, 18-hr saline (SAL), and 10-hr SAL infusions: Body weight and food intake were measured weekly. After 6 weeks, animals were killed; blood samples were taken for radioimmunoassay (RIA) of serum follicle-stimulating hormone (FSH) and prolactin (PRL); and terminal body, epididymal white adipose tissue (EPIWAT), and paired testis weights were recorded. Six-hour MEL infusions failed to induce short-day-type effects, regardless of the period (T) of the infusion cycle. In contrast, compared to SAL and 6-hr MEL infusions, 10-hr MEL resulted in decreases in body, EPIWAT, and testis weights in T 24, but not in T 36 or T 48. In T 18, testis, body, and EPIWAT mass were decreased, but not to the same extent as in T 24. Similarly, daily 18-hr MEL infusions (T24) were less effective as a short-day stimulus than were 10-hr MEL infusions. The effectiveness of 10-hr, but not 6-hr, MEL infusions in T 18 and T 24 is consistent with the duration hypothesis and argues against the circadian hypothesis. Neither hypothesis could have predicted that all infusion cycles of T greater than or equal to 36 hr, regardless of the infusion durations, would fail to elicit short-day-type responses. This outcome suggests a need for relatively frequent (T less than 36 hr) MEL stimulation in addition to the requirement for adequate duration of each MEL infusion.

Adipose Tissue↗

Germ cell control of testin production is inverse to that of other Sertoli cell products.

Recent studies have shown that germ cells can regulate testins, two newly identified Sertoli cell proteins that are associated with junctional complexes. To investigate this possibility, several parameters of Sertoli cell function were investigated over 2-120 days post exposure of the rat testes to x-rays (3 Grays). The irradiation-induced loss of spermatogonia resulted in a maturation-depletion process progressively affecting all germ cell classes. Testis weight began to decrease when the most numerous germ cell type (spermatids) began to decline. A complete or near complete recovery of spermatogenesis and of the testis weight had occurred by day 120 post irradiation. There was no significant change in FSH, epididymal androgen-binding protein, and tubule fluid levels during the first weeks after irradiation, when the seminiferious epithelium was depleted of spermatogonia and germ cells up to early spermatids. In contrast, when the number of the more mature forms of spermatids declined (between day 21 and 54), FSH rose and androgen-binding protein as well as fluid production declined. The subsequent recovery of these parameters was also highly correlated with the number of late spermatids. By contrast, testicular testin contents reacted to the depletion of germ cells with a biphasic increase; a doubling occurred when spermatogonia, spermatocytes, and early spermatids were absent (days 4-28), and a 7-fold rise occurred by day 37 when the number of late spermatids had decreased by 50%. By day 54, when the sperm counts had reached a nadir, testin contents had returned to levels corresponding to about four times the control levels; they progressively recovered thereafter. These observations support the postulate that germ cells negatively regulate testins. This possibility was investigated with in vitro experiments showing that addition of germ cell-conditioned medium to Sertoli cell monolayers inhibited testin secretion in a dose-dependent manner. In conclusion this study; 1) highlights the complex interplay between the various germ cell classes in the control of the Sertoli cell function in the adult testis; 2) establishes that germ cell effects may be opposite on different Sertoli cell products; 3) demonstrates that several classes of germ cells negatively control testicular testin contents; and 4) emphasizes the particular role of late spermatids in Sertoli cell regulation.

Androgen-Binding Protein↗

Changes in daily sperm production rate in rats under the influence of a potent antispermatogenic agent, CDRI 84/35.

CDRI 84/35, a potent nonsteroidal antispermatogenic agent, causes total sterility in rats by directly acting on germ cells while having no effect on Sertoli/Leydig cells. This study was conducted to evaluate the effect of the compound on gametogenic activity of testes and to identify stages of spermatogenesis that were affected. Adult male rats administered either compound 84/35 at minimum effective dose or estradiol (5 micrograms) or water only were killed on days 22, 41, and 64 of the treatment period to evaluate the effect on spermatid, spermatocyte, and spermatogonial stages, respectively. Daily sperm production (DSP) was measured employing a homogenization technique. Results showed a decline in testis weight and DSP with a drastic reduction (approximately 95%) in DSP in 84/35-treated rats on day 41 of the treatment period. Estradiol was more potent in reducing the testis weight; however, 84/35 had an edge over estradiol in reducing the DSP. After withdrawal of treatment for 120 days, a phenomenal recovery (> 90%) in DSP per gram parenchyma was noted in 84/35-treated animals. Results indicate a direct effect of estradiol on spermatogonia, whereas 84/35 seems to affect the spermatocyte stage.

Animals↗

Potential indicators of reproductive toxicity: testicular sperm production and epididymal sperm number, transit time, and motility in Fischer 344 rats.

Comprehensive assessment of the effects of chemicals on male reproductive functioning requires study of effects on spermatogenesis and the quality of spermatozoa produced. As potential indicators of toxic effects, we have examined sperm production rate (SPR) and epididymal sperm numbers, transit time, and motility (percentage motile cells and swimming speeds) in Fischer-344 (F-344) rats. Groups (n = 14 or 15) of rats at 6, 8, 10, 15, and 20 weeks of age were studied to investigate the suitability of these indicators for detecting toxic effects, and to establish the age at which this strain of rat sexually matures. Mean SPRs (10(6) sperm/rat/day), as measured by enumerating spermatids in testicular homogenates, were 2 +/- 3, 22 +/- 6, 41 +/- 7, 49 +/- 6, and 46 +/- 7 at 6, 8, 10, 15, and 20 weeks, respectively. Epididymal sperm numbers and the percentage of motile spermatozoa also increased with age. No correlation was found between SPR and either testis weight or epididymal sperm number at 10, 15, or 20 weeks of age. Our results indicate that male F-344 rats become sexually mature between 10 and 15 weeks of age, and that testis weight and epididymal sperm number are unreliable quantitative indicators of sperm production rate. The low variability among adult rats in SPR and some of the other indicators of testicular and epididymal functioning examined in this study suggest that they should be sensitive measures of male reproductive toxicity.

Age Factors↗

Photoperiod regulates testis cell apoptosis in Djungarian hamsters.

Reproductive activity in the Djungarian hamster is controlled by seasonal variations in day length. Exposure to long days stimulates testis development, while exposure to short days induces testis regression. We recently found that testis regression after gonadotropin deprivation in rats is associated with increases in apoptosis. Here we sought to determine whether or not apoptosis is associated with the testis regression and/or recrudescence that occurs naturally in seasonally breeding mammals. Newborn male hamsters were maintained on long days (16L:8D) until 3 wk of age before being transferred to short days (8L:16D). Following decreases in serum FSH within 3 days of exposure to short days, testis weight decreased by 52% at Day 10, reaching a 70% decrease after 21 days. Analysis of testis cell DNA fragmentation showed a 4.9-fold increase of low-molecular-weight DNA as early as 5 days after transfer to short days; this was followed by a time-dependent decrease. The observed increases in testis cell apoptosis were correlated with decreases in serum testosterone, but decreases in Leydig cell LH receptor content were delayed. In a second study, 6-wk-old hamsters with regressed testes due to a 3-wk exposure to short days were transferred back to long days. After increases in serum FSH within 3 days of photostimulation, a 2-fold elevation in testis weight was found at Day 5. The increase in testis weight was associated with a 65% decrease of testis apoptosis within 5 days of photostimulation. Also, increases in serum testosterone and LH receptor content were observed after 5 and 10 days of exposure to long days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Relationships among measures of testicular development and endocrine function in boars.

Nine blood samples were taken at 30-min intervals from 36 Landrace X Large White boars at each of eight ages (42, 56, 70, 84, 98, 112, 126 and 140 d). Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and estradiol-17 beta (E2) were quantified by radioimmunoassay procedures. The maximum concentration of LH and the age at maximum concentration were predicted for each boar. Variability of LH samples was described for each boar by the pooled within age variance among LH samples and by the number of LH peaks. Measurements of testicular development taken at 140 d of age included: in situ testis width and length, excised testis weights and histological traits of excised testes (seminiferous tubule diameter, percentage of tubules with a lumen and percentage of tubules with active spermatogenesis). Pooled within line correlations were calculated with data from boars selected for either high or low testis weight. Correlations among the testicular traits ranged from .45 to .88. Luteinizing hormone concentration (mean over all ages) was related to measures of testicular development (r = .24 to .49). Concentrations of LH from 42 to 84 d of age were more highly correlated with testicular traits than were the concentrations from 98 to 140 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

Alleviation of pre-exposure of mouse brain with low-dose 12C6+ ion or 60Co gamma-ray on male reproductive endocrine damages induced by subsequent high-dose irradiation.

Irradiation has been widely reported to damage organisms by attacking on proteins, nucleic acid and lipids in cells. However, radiation hormesis after low-dose irradiation has become the focus of research in radiobiology in recent years. To investigate the effects of pre-exposure of mouse brain with low-dose (12)C6+ ion or 60Co gamma (gamma)-ray on male reproductive endocrine capacity induced by subsequent high-dose irradiation, the brains of the B6C3F1 hybrid strain male mice were irradiated with 0.05 Gy of (12)C6+ ion or 60Co gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy as challenging irradiation dose at 4 h after pre-exposure. Serum pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), testosterone, testis weight, sperm count and shape were measured on the 35th day after irradiation. The results showed that there was a significant reduction in the levels of serum FSH, LH, testosterone, testis weight and sperm count, and a significant increase in sperm abnormalities by irradiation of the mouse brain with 2 Gy of (12)C6+ ion or 60Co gamma-ray. Moreover, the effects were more obvious in the group irradiated by (12)C6+ ion than in that irradiated by 60Co gamma-ray. Pre-exposure with low-dose (12)C6+ ion or 60Co gamma-ray significantly alleviated the harmful effects induced by a subsequent high-dose irradiation.

Animals↗

Differential effects of FSH and testosterone on the maintenance of spermatogenesis in the adult hypophysectomized rat.

In order to clarify further the role of FSH in the maintenance of spermatogenesis, adult rats were treated with purified human FSH (2 X 5 IU/day per rat), testosterone (1.5 cm silicone elastomer implant) or a combination of both hormones for 2 weeks following hypophysectomy. After hypophysectomy alone, no elongate spermatids were observed and the numbers of pachytene spermatocytes and round spermatids observed were reduced when compared with untreated controls. Testosterone supplementation alone qualitatively maintained the formation of elongate spermatids in most seminiferous tubules, whilst in FSH-treated rats increased numbers of round spermatids and pachytene spermatocytes were observed when compared with hypophysectomized animals. Formation of elongate spermatids, however, did not occur under FSH treatment alone. A combination of FSH and testosterone treatment maintained spermatogenesis in an almost quantitative fashion. Numbers of pachytene spermatocytes and round spermatids were maintained at about 80% of levels seen in intact control animals. Treatment with FSH or testosterone alone maintained testis weights at significantly higher levels than those seen in hypophysectomized controls (FSH, 0.79 +/- 0.05 g; testosterone, 0.81 +/- 0.07 g; hypophysectomized, 0.50 +/- 0.04 g). Animals treated with FSH and testosterone showed testis weights 20% below control values (1.22 +/- 0.05 vs 1.51 +/- 0.06 g; P less than 0.05). No increases in intratesticular or intratubular androgen concentrations or in testosterone: dihydrotestosterone ratios were observed in any of the hormone-treated groups when compared with hypophysectomized controls. In all hypophysectomized animals testicular androgen concentrations were reduced to less than 5% of control values. The results obtained in this study suggest that FSH is involved in the maintenance of spermatogenesis in the adult rat and that the effects of FSH are not mediated through changes in intratesticular androgens. Low levels of testosterone in combination with FSH can almost quantitatively maintain spermatogenesis in adult rats.

Androgens↗

A comparison of the effects of bilateral efferent duct ligation and of partial epididymectomy on the testes of rats.

Testis weight as a percentage of body weight did not change following bilateral ligation of the efferent ducts (EDL) close to the epididymis, whereas following removal of part of the epididymis between the site of ligation and a point close to the junction between the caput and corpus (PCE), testis weight first rose linearly until Day 4 and then showed an exponential decrease between Days 4 and 28. After EDL, the perimeter of the seminiferous tubules rose for the first 7 days and then remained elevated, whereas after PCE, there was a linear decrease between Days 4 and 28. Following EDL, the percentage of altered and degenerated tubular cross-sections rose to about 30% and 10%, respectively, during the first 7 days after operation and then remained constant; after PCE, the percentage of altered tubules reached a maximum of 54% by 4 days and then fell, whereas the percentage of degenerated tubules continued to rise to 95% by 28 days. It would appear that all the effects of removal of a portion of the epididymis cannot be explained by blockage of the excurrent ducts, and a specific endocrine effect of the epididymis on the testis is proposed.

Animals↗