Hemicastrated Rats Research Articles

Androgenic effects of the aqueous extract of Pycnanthus angolensis (Welw.) Warb. (Myristicaceae) wood in hemicastrated male Wistar rats

Background: Androgen deficiency is the most common disorder of reproductive function and can lead to male sexual disorders. Objective: This study was designed to evaluate the androgenic effects of Pycnanthus angolensis (Welw.) Warb in hemicastrated rats. Materials and methods: Forty-two male rats were divided into 6 groups of 7 rats each, including a group of uncastrated rats that received distilled water (10 ml/kg); a group of castrated rats that received 10 ml/kg of distilled water; a group of castrated rats that received testosterone enanthate (3 mg/kg BW) per week intramuscularly; and 3 groups of castrated rats that received 43, 86 and 172 mg/kg of the aqueous extract of Pycnanthus angolensis, respectively. After 14 days of oral treatment, the rats were killed by decapitation. The blood was collected and the androgen-dependent organs were collected for histological sectioning, and biochemical analysis. The tail of the epididymis was used to assess sperm quality. Results: Treatment with the aqueous extract at doses of 43 and 86 mg/kg, significantly improved the sexual behavior of castrated rats, with increases of 25.92% and 22.74% intromissions frequency, and 67.06% and 56.46% mount frequency, compared to those in the castrated rats which did not receive any treatment. The extract also enhanced sperm quality in castrated rats. Both doses also significantly increased serum testosterone levels with rates of 45.07% and 49.00%, respectively; compared to those in the negative control group. Conclusion: In view of the aforesaid results, Pycnanthus angolensis (Welw.) Warb could be considered as a promising natural agent in hypogonadism management. Keywords: Androgen, hypogonadism, castrated male rats, unilateral castration, Pycnanthus angolensis (Welw.) Warb

Effect of Unilateral Iatrogenic Testicular Torsion on the Contralateral Testis in Rats: Prepubertal and Postpubertal.

The present study was conducted to investigate the influence of hemicastration and age at hemicastration on the contralateral testis weight and function/testosterone production. 64 Wistar-derived male rats were divided randomly into 4 groups. Group 1 was named immature intervention, group 2 immaturecontrol, group 3 mature intervention, and group 4mature control. In group 1, rats were hemicastrated at 30 days of age (prepubertal). In group 2, sham surgery (midscrotal incision) was per-formed atthe same age. In group 3, rats were hemicastrated at 70 days of age (postpubertal) and in group 4,sham surgery was done at the same age. Twenty days after the first surgery, contralateral orchiectomy wasperformed in intervention groups and controls underwent random orchiectomy (left or right). Blood sampling for evaluation of serum testosterone was performed just before second surgery. The mean testis weight (1692 ± 26.7 in group 1 versus 1375 ± 39.7 in group 2; P < .001 and 1760 ± 26.6 in group 3 versus 1425 ± 44.9 in Group 4; P < .001) and the mean testicular weight (mg) per 100 g of body weight (735.8 ± 82.3 in group 1 versus 634.8 ± 84.8 in group 2; P = .005 and 652.4 ± 61.4 in group 3 versus 572.6 ± 97.7 in group 4; P = .03) were significantly greater in hemicastrated rats as to their controls. Also, these parameters was greater in prepubertal group than postpubertalhemicastrated rats. There was no appreciable difference in serum testosterone levels across the 4 groups (P = .77). Our research demonstrated that hemicastration results in compensatory hypertrophy of the remaining testis and it decreases as the animals age. Hemicastration does not lead to reduction in serum testosterone levels and the remaining testis can retrieve a normal serum testosterone level.

Effect of vitamin E on oxidative stress in the contralateral testis of neonatal and pubertal hemicastrated rats

ObjectiveTo evaluate whether the antioxidant vitamin E can prevent the harmful effects of reactive oxidative stress (ROS) that occur during compensatory testicular hypertrophy (CTH). Materials and methodsThirty Sprague–Dawley rats were divided into six equal groups: neonatal hemicastrated vitamin E (NH_Vit E/NH) and sham surgical controls (NC), and pubertal hemicastrated vitamin E (PH_Vit E/PH) and sham surgical controls (PC). Vitamin E was administered orally to the NH_Vit E and PH_Vit E groups three times a week from week 3–12 prior to sacrifice. Antioxidant enzymes were measured in testis samples from each animal. ResultsDifferences in superoxide dismutase activity were observed between the NH (21.04 ± 0.48) and NH_Vit E (22.62 ± 0.64) groups (P = 0.008); the PH (20.59 ± 0.11) and PC (20.91 ± 0.20) groups (P = 0.032); and the PH (20.59 ± 0.11) and PH_Vit E (22.32 ± 1.01) groups (P = 0.008). Thiobarbituric acid-reactive substance in the PH and PH_Vit E groups was 0.097 ± 0.022 and 0.036 ± 0.004 (P = 0.008), respectively; and in the NH and NH_Vit E groups it was 0.135 ± 0.02 and 0.039 ± 0.003 (P = 0.008), respectively. ConclusionsThese results suggest that CTH is not associated with reducing oxidative injury, nor does it prevent ROS-induced cell damage. However, administration of vitamin E does reduce oxidative injury and prevent ROS-induced cell damage in a hemicastrated rat model.

Effect of Fructus Corni polysaccharides on damaged sexualfunction of male rats

To observe the effects of Fructus Corni polysaccharides (FCP) on sexual function of hemicastrated rats. 70 male SD rats are randomly divided into 7 groups with their right testis extirpated except for normal control group. Normal control group and negative control group are given saline (ig) while positive control group are injected hypodermically testosterone propionate at dose of 2 mg x kg(-1) x d(-1). FCP control groups are given FCP separately at dose of 10, 50, 100, 150 mg x kg(-1) x d(-1) (ig). Mating test and erective test are observed. The levels of serum sex hormone T, LSH, FSH, E2 are detected with the Radioimmunoassay (RIA). Incubation period of penis erection and mounting are shortened in FCP control groups and positive control group, and the percentage of mounting rats is increased. The level of serum sex hormone T is increased, but estradiol level is reduced. The organ coefficient of foreskin gland and seminal vesicle-prostate gland as well as sperm count and vigor are increased significantly (P < 0.01 or P < 0.05). FCP can increase the sexual function of hemicastrated rats. The mechanism is probably through adjustment of the hypothalamus-pituitary-gonadal axis.

The effect of neonatal vasectomy on testicular function.

The effect of unilateral or bilateral vasectomy on testicular weight and basal testosterone production in vitro was studied in neonatal intact and hemicastrated rats. Five days after right-side vasectomy ipsilateral to vasectomy testicular weight increased, and basal testosterone production decreased. Ten days postvasectomy the changes were opposite, and affected both testes. In hemicastrated animals hemivasectomy did not interfere with compensatory hypertrophy but induced a significant decrease in basal testosterone production. The data of the present study suggest that in neonatal animals intact ductus deferens bundle(s) is also required for the full control of testicular weight and basal testosterone secretion.

Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis

Testosterone secretion by Leydig cells is affected by interleukin-1β (IL-1β). The aim of the present study was to investigate whether partial denervation of the testis or local administration of a serotonin (5-HT) receptor antagonist could alter the changes in testicular steoidogenesis induced by IL-1β. Intratesticular administration of IL-1β was combined with vasectomy or local injection of ketanserin (5-HT type 2 receptor antagonist) in immature hemicastrated rats and the effect of the interventions on testicular steroidogenesis was studied. One day after treatment with local injection of IL-1β induced a significant rise in testosterone secretion that could be prevented by vasectomy (that also means transection of the inferior spermatic nerve). In a model in which neither IL-1β nor ketanserin interfered with steroidogenesis, administration of the receptor antagonist just prior to IL-1β treatment significantly reduced testosterone secretion. Data indicate interaction between testicular nerves and IL-1β action and interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion.

Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H 2O 2-induced DNA damage in mouse testicular cells and beneficial effect on sexual behavior and reproductive function of hemicastrated rats

Lycium barbarum, a famous Chinese medicinal herb, has a long history of use as a traditional remedy for male infertility. Polysaccharides are the most important functional constituent in L. barbarum fruits. We systematically investigated the effect of L. barbarum polysaccharides (LBP) on rat testis damage induced by a physical factor (43 °C heat exposure), on DNA damage of mouse testicular cells induced by a chemical factor (H 2O 2), and on sexual behavior and reproductive function of hemicastrated male rats. The results showed that LBP provided a protective effect against the testicular tissue damage induced by heat exposure. When compared with negative control, a suitable concentration of LBP significantly increased testis and epididymis weights, improved superoxide dismutase (SOD) activity, and raised sexual hormone levels in the damaged rat testes. LBP had a dose-dependent protective effect against DNA oxidative damage of mouse testicular cells induced by H 2O 2. LBP improved the copulatory performance and reproductive function of hemicastrated male rats, such as shortened penis erection latency and mount latency, regulated secretion of sexual hormones and increased hormone levels, raised accessory sexual organ weights, and improved sperm quantity and quality. The present findings support the folk reputation of L. barbarum fruits as an aphrodisiac and fertility-facilitating agent, and provide scientific evidence for a basis for the extensive use of L. barbarum fruits as a traditional remedy for male infertility in China.

Effect of edifenphos on compensatory ovarian hypertrophy, follicular kinetics and estrous cycle in hemicastrated rats.

Edifenphos, an organophosphate fungicide, was administered to hemicastrated albino rats intraperitoneally for 15 consecutive days in graded doses of 2 to 8 mg/kg body weight. In the oil-treated hemicastrated control group, ovarian weight and total number of healthy and atretic follicles were significantly higher than the same parameters in sham-operated control animals, and the estrous cycle was normal. Treatment with 2, 4, 6, or 8 mg/kg/d edifenphos significantly decreased ovarian weight (-6.75, -9.79, -18.71, and -34.13 percent, respectively) below that of the controls. Although treatment with 2 or 4 mg/kg/d edifenphos did not change the number of healthy and atretic follicles, a dosage of 4 mg/kg/d significantly decreased the number and duration of estrous cycles. Treatment with 6 or 8 mg/kg/d, however, significantly decreased the number and duration of different phases of the estrous cycle as well. In all treated groups, the weights of the thymus and uterus were significantly reduced when compared with those of hemicastrated oil-treated control animals.

Testicular denervation-induced nuclear changes in Leydig cell of hemicastrated adult rats

In the adult male rat, hemicastration (HC) induces a remarkable enhancement of testosterone secretion from the Leydig cells of the remaining testis. We have observed previously that the denervation of inferior spermatic nerves inhibits this enhancement. The present experiments were designed to assess morphometrically whether or not this change had a cytological correlate in Leydig cells. At least five testes from each group (denervation plus HC, sham denervation plus HC and intact rats) were prepared for both optical and electron microscopy studies. The results showed that after 24 h of denervation, the nuclear volume of the Leydig cells in denervation plus HC rats was smaller ( p < 0.01) than those of sham denervation plus HC (196.56 ± 16.53 vs. 280.71 ± 13.37), whereas both the volume density of heterochromatin (19.84 ± 3.14 vs. 10.03 ± 2.47%) and the heterochromatin index (expressed as periphery heterochromatin area divided by nuclear perimeter, 0.149 ± 0.046 vs. 0.094 ± 0.026) were significantly ( p < 0.01) higher in denervation plus HC rats than in its sham groups. No changes in Leydig cell numbers or cytoplasmic organelles were detected. The results suggest that some nuclear and heterochromatin-associated cellular activity might be inhibited by testicular denervation in hemicastrated rats.

Local Effect of PACAP and VIP on Testicular Function in Immature and Adult Rats

Csaba, Zs., V. Csernus, and I. Gerendai. Local effect of PACAP and VIP on testicular function in immature and adult rats. Peptides 18(10) 1561–1567, 1997.—PACAP, VIP, anti-PACAP and anti-VIP antisera were injected intratesticularly. In 9-day-old hemicastrated rats PACAP or VIP decreased basal testosterone secretion. In 22-day-old hemicastrates VIP but not PACAP reduced compensatory testicular hypertrophy, however, neither PACAP nor VIP altered steroidogenesis. Anti-VIP antiserum to this age group increased testosterone production and enhanced compensatory testicular hypertrophy. In adult hemicastrates neither the peptides nor the antisera influenced steroidogenesis. Neither in immatures nor in adults treatment of both testes with PACAP or VIP had any effect. Data indicate that both PACAP and VIP might exert a local action on testicular steroidogenesis, on compensatory testicular hypertrophy, and these effects are age-dependent.

Testicular and serum levels of inhibin and expression of inhibin subunit mRNAs are differentially affected by hemicastration in rats of various ages.

Age-related short-term effects of hemicastration on testicular weight, serum FSH, immunoreactive inhibin, LH and testosterone, testicular levels of inhibin subunit mRNA expression, and bioactive and immunoreactive inhibin were studied in rats of 8, 15 and 22 days of age. Hemicastration led to an increased weight of the remaining testis after 24 h in 8- and 15-day-old rats, but not in 22-day-old rats. Serum FSH levels were elevated in all hemicastrated rats after 8 h. However, serum immunoreactive inhibin levels were decreased only after 72 h in 8-day-old rats and after 24 h in 15- and 22-day-old rats. Inhibin alpha-subunit mRNA expression was increased in the testes of hemicastrated rats of 8 and 15 days of age, whereas inhibin beta B-subunit mRNA expression was elevated in the testes of 15-day-old rats but not in those of 8- and 22-day-old rats. The increase in alpha-subunit mRNA content per testis was caused by an increased concentration and increased testicular weight, whereas the increase in beta B-subunit mRNA in the remaining testis parallelled the increased testicular weight, indicating that different mechanisms play a role in the regulation of these mRNAs. In 22-day-old rats, a transiently decreased expression of inhibin beta B-subunit mRNA was observed 8 h after hemicastration.(ABSTRACT TRUNCATED AT 250 WORDS)

Duration of the cycle of the seminiferous epithelium, estimated by the 5-bromodeoxyuridine technique, in laboratory and feral rats

The stability of the duration of the cycle of the seminiferous epithelium was determined by investigating incorporation of 5-bromodeoxyuridine into S-phase germ cells of normal and hemicastrated standard laboratory rats (Sprague-Dawley) and feral Brown/Norway rats (Rattus norvegicus). Feral rats were trapped on farms in the surroundings of Münster. The duration of the cycle of the seminiferous epithelium, determined at intervals of 12 days (3 h versus 12 days 3 h after 5-bromodeoxyuridine injection), was remarkably constant and similar in intact laboratory rats (12.49 +/- 0.05 days, n = 13, mean +/- SEM) and feral rats (12.44 +/- 0.06 days, n = 8). In hemicastrated laboratory and feral rats the duration of the cycle was similar to that in intact animals, indicating that hemicastration did not influence the kinetics of the seminiferous epithelium cycle. However, the coefficients of variation of the estimated duration of the cycle of the seminiferous epithelium were at least three times lower in hemicastrated rats (one testis from the same animal serving as reference point) compared with that of intact rats (the reference point based on the average staining frequency at 3 h). Overall, no significant differences between laboratory and feral rats could be observed with regard to testis weight and serum concentrations of FSH and testosterone. The number of cells per testis, determined by flow cytometry, was similar in laboratory and feral rats, except for a slight but significant difference in the haploid:tetraploid cell ratio (6.3 +/- 0.2 versus 7.5 +/- 0.3, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of follicular fluid administration on serum bioactive and immunoactive FSH concentrations and compensatory testicular hypertrophy in hemicastrated prepubertal rats

In prepubertal rats, removal of one testis (hemicastration) results in increased FSH secretion and a compensatory hypertrophy of the remaining testis. To determine whether these two events are related, testis weight was measured after inhibin-rich porcine follicular fluid (FF) was administered to hemicastrated rats twice daily for 14 days to block the compensatory rise in FSH. After hemicastration, serum immunoreactive FSH concentration approximately doubled and testis weight was increased by approximately 60%. A slight but non-significant increase in serum bioactive FSH was also observed. Treatment of hemicastrated rats with FF completely prevented the increase in serum FSH and compensatory growth of the remaining testis, whereas concomitant treatment of these animals with ovine FSH reversed the inhibitory effects of FF. Serum inhibin concentrations were determined using two radioimmunoassay (RIA) systems; one assay utilized an antiserum generated against intact bovine inhibin and the other was directed against an alpha-inhibin fragment. Both assays showed a decline in inhibin levels following hemicastration. In addition, increases in serum inhibin concentrations were observed following FF administration in both RIAs; however, the relative increase in inhibin over levels in hemicastrated rats was greater using the intact inhibin assay. In summary, these data suggest that the increase in FSH concentrations after hemicastration is related to a reduction in inhibin levels, and that this FSH rise is a primary signal for initiating compensatory testicular hypertrophy in prepubertal rats. Furthermore, exogenous FSH administration overcame the inhibitory effects of FF on the testes, suggesting that inhibin may not act directly at the gonadal level.

Effects of follicular fluid administration on serum bioactive and immunoreactive FSH concentrations and compensatory testosterone secretion in hemicastrated adult rats.

In adult rats, removal of one testis (hemicastration) results in an elevation of serum follicle-stimulating hormone (FSH) concentrations and a compensation in testosterone secretion by the remaining testis without a corresponding increase in testis size. To determine whether changes in FSH secretion and compensatory androgen production are related, serum testosterone concentrations were measured after inhibin-rich porcine follicular fluid was administered twice daily for 4 days to block the hemicastration-induced rise in FSH. Both serum immunoreactive FSH (immuno-FSH) and bioactive FSH (bio-FSH) concentrations were increased 4 days after hemicastration. The significant increase in serum immuno-FSH in hemicastrated animals was prevented by follicular fluid administration, whereas the serum bio-FSH activity and biologic to immunologic (B/I) ratios were increased in follicular fluid-treated animals. The follicular fluid-induced reduction in serum immuno-FSH had no effect on serum testosterone secretion in hemicastrated rats. Serum inhibin concentrations were reduced 27% in hemicastrated rats compared with intact controls, while administration of exogenous follicular fluid increased serum inhibin concentrations. An elevation in serum immuno-FSH secretion after hemicastration apparently is not required for the compensatory testosterone response. However, the observation of increased bio-FSH in hemicastrated and follicular fluid-treated animals raises questions about the importance of FSH quality (bioactivity), rather than quantity, for controlling testicular steroidogenic activity.

Onset of Compensatory Hypertrophy of Interstitial Tissue and Leydig Cells in Rats Hemicastrated around the Time of Puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.

Intratesticular injection of [ d-Met 2-Pro 5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [ d-Met 2-Pro 5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1–0.3 μg/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 μg/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.

Differential effects of a unilateral hypothalamic lesion on ovulation and compensatory ovarian hypertrophy in hemiovariectomized adult rats

The effects were analysed of a unilateral lesion in the anterior or medial hypothalamus made on the day of oestrus on right or left hemicastrated rats. On the day of oestrus after two consecutive oestrous cycles of the same length, the ovulation rate in rats with lesions in the anterior left hypothalamus was lower than in control hemicastrated animals (5/16 vs 18/20; P less than 0.01), and normal in those rats with lesions in the right side (14/18). None of the animals with lesions in the left side of the anterior hypothalamus and with the left ovary in situ ovulated (0/7), but 5/9 with the right ovary in situ did ovulate (P less than 0.05). Lesions on either side of the medial hypothalamus did not modify ovulation rate. Compensatory ovulation was reduced in those animals with lesions in the right anterior hypothalamus and with the right ovary in situ. Lesions in either side of the medial hypothalamus reduced compensatory ovulation. Lesions in the right side of the anterior and medial hypothalamus increased compensatory ovarian hypertrophy in the left ovary and decreased it in the right ovary. Lesions in the left side of the anterior hypothalamus increased compensatory ovarian hypertrophy in the left ovary only, whereas lesions in the left side of the medial hypothalamus reduced compensatory ovarian hypertrophy in the right ovary. The results suggest that the information arising in each side of the anterior and medial hypothalamus plays different roles in the ipsi-and contralateral ovary, when either the left or the right ovary is absent.

Influence of neonatal hemicastration on in-vitro secretion of inhibin, gonadotrophins and testicular steroids in male rats

Pituitary secretion of FSH in male animals is regulated, at least partly, by a protein hormone, inhibin, which is produced by Sertoli cells in the testes. To establish at which age the role of testicular inhibin in the regulation of FSH secretion becomes apparent, groups of male rats were hemicastrated or sham-operated on day 1 of life and pituitary and testicular function were investigated in vitro at 21, 42 or 63 days of age. Testis weights were increased in hemicastrated rats at all ages studied. Peripheral concentrations of gonadotrophins generally showed a good correlation with the concentrations of FSH and LH measured in the medium of hemipituitary glands which were incubated in vitro at 37 degrees C in the absence or presence of LH-releasing hormone. Peripheral testosterone concentrations in hemicastrated animals were not significantly different from those in sham-operated rats at all ages studied. Steroid production by Leydig cells in vitro was not significantly influenced by hemicastration. The secretion of inhibin by Sertoli cells from 21-day-old hemicastrated rats was decreased while Sertoli cells from 42- and 63-day-old hemicastrated animals secreted slightly but not significantly more inhibin than Sertoli cells from sham-operated rats. It is concluded that although compensatory increases of testosterone and inhibin production at later ages make it difficult to draw conclusions about the relative importance of inhibin in the feedback regulation of FSH secretion at different ages, it is likely that inhibin plays a role in the feedback of FSH in immature, rather than in mature male rats.

Evidence that the anterior hypothalamus contributes to the control of tonic secretion of follicle-stimulating hormone in the female rat.

A horizontal knife cut was placed between the dorsal anterior hypothalamic area (DAHA) and the medial basal hypothalamus to examine the role of the DAHA in the selective secretion of follicle-stimulating hormone (FSH) following unilateral ovariectomy (ULO) and bilateral ovariectomy of the rat. Complete cuts markedly attenuated the increase in FSH observed 8 h after ULO, whereas incomplete or sham cuts did not. Concentration of luteinizing hormone (LH) did not increase in any group. These cuts also blocked the prolongation of estrous FSH secretion observed in long-term hemicastrated rats, since FSH levels on estrus were significantly lower in rats with complete cuts than in those rats given sham or incomplete cuts. In contrast, neural surgery had no effect on proestrous FSH concentrations. Finally, when FSH levels were monitored 2 days after bilateral ovariectomy, the postcastration rise in FSH was not altered by any neurosurgical procedures. These results support the hypothesis that a neural system originating in, or passing through, the DAHA is necessary for the selective increase in FSH following ULO.

Nuclear androgen binding sites in the male rat—III. Late spermatids and spermatozoa in the testis, with an introduction to epididymal spermatozoa

Nuclear androgen binding sites were examined in late spermatids (stages 12–19) which resisted sonication of homogenized testes of mature male rats. The measurement of unoccupied binding sites in salt extract of purified spermatid heads by nuclear exchange at — 10°C was developed and validated. As in the prostate, unoccupied nuclear androgen binding sites in sonicated testes were in low concentration, were not artefactual, and could be occupied both in vivo and in virto by exogenous androgens, and uniquely in hemicastrated rats by endogenously compensated androgens in the remaining testis. The properties of occupied binding sites in salt extract of purified spermatid heads (measured by nuclear exchange at 4°C for 48 or more hours with 5nM [ 3H]dihydrotestosterone) were almost identical to those of occupied binding sites in nuclei of the ventral prostate, except for their concentration. However, levels of specific binding activity approaching 50 fmol/mg DNA could be expected in salt extract of spermatid pellets, by use of a sulfhydryl reducing agent (dithiothreitol) prior to salt extraction, a protease inhibitor (phenylmethylsulfonyl fluoride) in all buffers, and optimization of the sonication protocol. Nuclear androgen binding sites of sonicated epididymal spermatozoa, collected by retrograde perfusion of the cauda epididymidis, were found to be completely salt-resistant. These binding proteins could be extracted by 0.4 M KG if dithiothreitol and dihydrotestosterone were incorporated into the sonication buffer, if phenylmethylsulfonyl fluoride was added to all buffers, and if the purified epididymal sperm pellet was treated with sarkosyl, a non-ionic detergent, just before salt extraction. The salt extract of epididymal spermatozoa which were treated as described above contained two binding components: a soluble form which was eluted from hydroxylapatite by increasing concentrations of phosphate buffers, and a non-soluble form, free of DNA, which remained in the hydroxylapatite column, and which contained most of the androgen binding sites. Affinity ( K d ) of dihydrotestosterone to the soluble and insoluble fractions of the steroid-binding protein complex was determined to be 0.7 and 0.1 nM, respectively. Salt-resistance of binding proteins in germ cells was shown to develop significantly in the last stages of spermiogenesis.