Suppression of testicular maturation and fertility following androgen administration to neonatal male rats

Administration of supraphysiologic doses of androgen to male rats within the first few neonatal days markedly suppresses subsequent testicular maturation; this effect diminishes as androgen is injected on succeeding postnatal days. Testosterone propionate (TP) administered neonatally at dosages up to 3.5 mg appreciably diminished postnatal testicular growth; postpubertal androgen secretion, as assessed by accessory sex organ weights and serum testosterone concentrations and as reflected by a castrationlike developmental pattern of the hepatic enzyme, histidase; spermatogenesis; and fertility. Beyond three mo of age testicular growth rates and androgen secretion--but not fertility--tended to be restored. These effects of neonatal androgen do not require aromatization to estrogen; indeed 5 alpha-dihydrotestosterone elicited more profound testicular suppression than TP, which was sustained until at least 100 days of age. Testes of neonatally androgenized rats were capable of responding to gonadotropins administered at three wk of age with increases in weight and androgen secretion. These findings suggest that a developmental event, suppressible by pharmacologic doses of androgen, occurs at a nontesticular site during the first few post partum days in the male rat; this event programs subsequent testicular maturation.     

Androgen binding protein, sperm production, semen output and LH and testosterone profiles in young postpubertal bulls

An androgen binding protein (ABP), which binds 5alpha-dihydrotestosterone with high affinity (Ka = 0.3 x 10(9) M(-1)), has been demonstrated in testicular and epididymal cytosols of 5 young post pubertal bulls (15-17 months old) of the Montbeliarde dairy breed. Simultaneously, daily sperm production (DSP), semen output and plasma LH and testosterone concentrations (from frequent samplings) were determined. ABP levels were 21 fmoles/mg protein in testis and 59, 22 and 43 fmoles/mg, respectively, in caput, corpus and cauda epididymis. Mean DSP, per gram of testis, was 16.6 x 10(6) spermatozoa, and the mean sperm output was approximately 1.5 x 10(9) spermatozoa per ejaculate. Mean LH and testosterone levels were 1.5 ng/ml and 2.1 ng/ml, respectively. One bull (882) was clearly distinguishable from the others, in showing higher ABP and testosterone levels together with a lower daily sperm production. Results of this study may (1) suggest a physiological role of ABP in sperm epididymal maturation and (2) give a new parameter in the evaluation of individual bulls testicular function.     

La surexpression de l'androgen-binding protein (ABP) provoque des modifications morphologiques et fonctionnelles dans le testicule de souris

The Sertoti cells (SC) of many species produce an androgen-binding protein (ABP) which is secreted into both the blood and lumen of the seminiferous tubule. In the latter, it is transported to the epididymis where is taken up by epithelial cells, and is thought to play a role in sperm maturation. In view of the importance of ABP, we thought it would be pertinent to make several transgenic mice (TM) lines bearing the rABP gene to unravel its role in male reproductive physiology. A 5.5 Kb rat genomic DNA clone was microinjected into the pronucleus of fertilized mouse ova which were subsequently implanted into the oviduct of pseudopregnant CD-1 female mice. Detection of TM was performed by Southern Blot and PCR analysis using respectively, a 32p labeled rABP cDNA probe and oligonucleotides recognizing exons 1 and 7 of rABP gene. Chromosomic localization of the transgene was carried out by fluorescent in situ hybridization (FISH) in metaphasic cells obtained from bone marrow of TM. rABP expression was analyzed by Northern blot and RT-PCR techniques in most tissues of heterozigote TM. In the testis, specific cell expression was determined by in situ hybridization (ISH) and protein localization by immunohistochemistry. ABP-binding activity was performed by the carbon dextran method and analysis of protein internalization by autohistoradiographic detection of a (3H) testosterone-ABP complex. DNA fragmentation was investigated by the TUNEL technique and by electrophoresis of total genomic DNA. Testicular and epididymal morphology was studied by light and electron microscopy. Two offspring carrying the rABP gene were identified by Southern blotting, and two lines of mice (designated ABP7 and ABP24) were generated by selective breeding of the male founders with normal B6D2F1 females. FISH analysis demonstrated a different chromosomal localization of the transgene in both lines. Both rABP transgenic pedigrees presented reduced fertility. Northern blot and RT-PCR studies showed overexpression of rABP mRNA in the testis. ovary and uterus in ABP24 and ABP7 transgenic lines. rABP mRNA was appropriately expressed in SC as demonstrated by ISH. DHT-sH binding of testicular homogenates was increased 10 fold in TM compared to controls. In adult testis of TM, some seminiferous tubules showed disorganization of the epithelium, increased number of SC, presence of vacuoles, germ cell meiotic arrest and germ cell degeneration. DNA fragmentation was demonstrated in germ cells during meiosis. rABP protein was localized in the intersticial space, and into some tubules, in SC and germ cells at different steps of maturation. rABP internalization was strongly increased in both germ and epididymal cells in TM. The present results reinforce the increasing evidence of the role of ABP during spermatogenesis, even though further experiments are required to unravel its definitive implication in testicular and epidididymal homeostasis.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Delay in the age of balano-preputial skinfold cleavage and alterations in serum profiles of testosterone, 5 alpha-androstane-3 alpha,17 beta-diol, and gonadotropins in adult rats treated during puberty with luteinizing hormone releasing hormone

Previous work has shown that chronic treatment of intact, immature male rats with luteinizing hormone releasing hormone (LHRH) decreases sex accessory gland weights and results in retardation of the normal developmental increase in the ratio of serum testosterone (T)/5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Diol) via an apparent enhancement of testicular 5 alpha-reductase or 3 alpha-hydroxysteroid oxidoreductase activities. In the present work, androgen dependent balano-preputial skinfold cleavage was significantly delayed by approximately one week in intact, immature male rats which were treated daily for two weeks with either 1.0 micrograms, 2.5 micrograms or 5.0 micrograms of LHRH during a discrete phase of pubertal development (28-41 days of age). In intact, adult (62 day old) animals which received LHRH treatments during pubertal development, serum T concentrations and sex accessory gland weights were reduced compared to control animal values. Serum 3 alpha-Diol content in the adult rats was either unaltered or increased significantly depending on the LHRH dosage employed during sexual development. Serum luteinizing hormone concentrations were not different between control and LHRH-pretreated adult rats whereas the highest dosage of LHRH employed (5.0 micrograms) during puberty resulted in a significant elevation of adult serum follicle stimulating hormone levels. It is suggested that chronic LHRH treatment of the male rat during puberty results in a perturbation in testicular androgen biosynthetic activities and an impairment of pituitary-testicular hormone feedback mechanisms which persist at least through early adulthood.     

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.     

Alteration of free sulphydryl content of rat sperm heads by suppression of intratesticular testosterone

Subcutaneous injections of testosterone propionate to adult male rats at a dose of 2.5 or 10 mg/kg body weight, 3 times per week for 7 weeks, resulted in a 75% reduction in serum LH and more than 50% reduction in intratesticular testosterone concentration, but serum FSH levels remained unchanged. The free -SH content, measured as iodo[14C]acetamide binding, increased by 70-100% in testicular sperm heads after suppression of testicular testosterone, and by 25-30% in caput epididymal sperm heads but was decreased by 70-80% in cauda epididymal sperm heads. These results demonstrate an alteration in the oxidative state of sperm nuclear basic proteins, suggesting incomplete nuclear maturation. These changes may be specific for the suppression of intratesticular testosterone, thus illustrating the androgen dependency of sperm head maturation. The contrast effects noted between the iodo[14C]iodoacetamide binding by the caput and the cauda epididymal sperm heads indicate that testosterone propionate treatment may affect the mechanisms regulating the oxidation of the sulphydryl residues in sperm heads during epididymal transit. This alteration may not directly relate to the tissue androgen concentrations.     

The distribution of rat testibumin in the male reproductive tract

A glycoprotein, designated CMB-1, has been identified in media from Sertoli cell-enriched cultures that increases in concentration in response to follicle-stimulating hormone (FSH) and testosterone. Subsequent studies indicated that CMB-1 is immunologically related to albumin and alpha-fetoprotein and is concentrated in the luminal compartment of the testis in adult rats. Thus, CMB-1 was termed testibumin. The goal of the present study was to determine the concentrations of this protein in testes, epididymides, and serum of normal rats between 10 and 180 days of age and to compare them to rat androgen-binding protein (rABP). Testibumin concentration in rat testes increased with age and peaked at Day 60; thereafter, unlike rABP, its concentration declined, reaching a plateau by 150 days of age. Testibumin concentration in the epididymal compartment also increased with age and peaked at Day 90; thereafter, its concentration remained relatively unchanged. Unlike rABP, which accumulates in the caput epididymis, testibumin did not accumulate preferentially in any particular region of the epididymis. In spite of the marked changes of testibumin concentration in the male reproductive tract, the levels in blood remained relatively constant between 10 and 180 days of age. In adult male and female rats, the serum concentrations of testibumin were similar. Following orchiectomy, serum testibumin concentration decreased by 50% with an apparent t1/2 of approximately 8 h. The presence of immunoreactive macromolecules in other species that share epitopes with rat testibumin was also investigated. Material in human sera and extracts of human and monkey testes cross-reacts with rat testibumin. After [35S]methionine was added to the primary Sertoli cell-enriched cultures, anti-testibumin antiserum selectively immunoprecipitated a radiolabeled protein with the same electrophoretic mobility as purified testibumin on sodium dodecyl sulfate (SDS)-polyacrylamide gels. We conclude that 1) rat testibumin is synthesized and secreted by Sertoli cell-enriched cultures; 2) the relative concentrations and distribution of testibumin in testis, epididymis, and serum of the rat as a function of age are strikingly different from those of rABP; 3) rat testibumin shares epitopes with proteins in human serum and testicular extracts of monkey and man.     

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.     

α‐Lipoic acid inhibits testicular and epididymal oxidative damage and improves fertility efficacy in arsenic‐intoxicated rats

The present study evaluates the protective effect of α‐lipoic acid (LA) against arsenic‐induced testicular and epididymal oxidative damage in rats. Arsenic caused significant reduction in the reproductive organ weights, serum testosterone levels, testicular daily sperm count, epididymal sperm count, sperm motility, sperm viability, and sperm membrane integrity. Significant reduction in the activity levels of superoxide dismutase, catalase, and glutathione levels with a concomitant increase in the lipid peroxidation and protein carbonyl content in the testis and the cauda epididymis of arsenic‐exposed rats. Arsenic intoxication also enhanced the testicular caspase‐3 mRNA levels, disorganization of testicular and cauda epididymal architecture as well as increased arsenic content in the testis and the cauda epididymis of rats. Arsenic exposure also deteriorated fertility ability in male rats over controls. Conversely, α‐LA negated the testicular and cauda epididymal oxidative stress and restored the male reproductive health in arsenic‐exposed rats.     

Effects of FSH receptor deletion on epididymal tubules and sperm morphology, numbers, and motility

Follicle stimulating hormone (FSH) interacts with its cognate receptor (R) on Sertoli cells within the testis and plays an important role in the maintenance of spermatogenesis. Male FSH-R knockout (FORKO) mice show fewer Sertoli cells and many that are structurally abnormal and as a consequence fewer germ cells. Lower levels of serum testosterone (T) and androgen binding protein (ABP) also occur, along with reduced fertility. To assess the effects of FSH-R depletion as an outcome of testicular abnormalities, sperm from the cauda epididymidis were counted and examined ultrastructurally. As reduced fertility may also reflect changes to the epididymis, the secondary responses of the epididymis to lower T and ABP levels were also examined by comparing differences in sizes of epididymal tubules in various regions of FORKO and wild type (WT) mice. Sperm motility was evaluated in FORKO mice and compared to that of WT mice by computer assisted sperm analysis (CASA). Quantitatively, the data revealed that epithelial areas of the caput and corpus epididymidis were significantly smaller in FORKO mice compared to WT mice. Cauda epididymal sperm counts in FORKO mice were also much lower than in WT mice. This resulted in changes to 9 out of 14 sperm motility parameters, related mostly to velocity measures, which were significantly lower in the FORKO mice. The greatest change was observed relative to the percent static sperm, which was elevated by 20% in FORKO mice compared to controls. EM analyses revealed major changes to the structure of the heads and tails of cauda luminal sperm in FORKO mice. Taken together these data suggest a key role for the FSH receptor in maintaining Sertoli cells to sustain normal sperm numbers and proper shapes of their heads and tails. In addition, the shrinkage in epididymal epithelial areas observed in FORKO mice likely reflect direct and/or indirect changes in the functions of these cells and their role in promoting sperm motility, which is noticeably altered in FORKO mice.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.     

Androgen binding to cytosol prepared from epididymides of sexually mature castrated rabbits: evidence for a cytoplasmic receptor.

The presence of androgen-binding activity in cytosol prepared from the major anatomical segments (caput, corpus, and cauda) of the epididymis of castrated sexually mature rabbits has been demonstrated. A portion of this binding activity is likely to be the epididymal androgen receptor. When epididymal cytosol from adult castrated rabbits is analyzed on low-ionic strength (0.01 MKCl) sucrose gradients, two peaks of macromolecular binding could be detected, one congruent to 4.6S and one congruent to 8S. On gradients containing 1.0 M KCl, only one sedimenting form congruent to 4.6S could be demonstrated, suggesting that the 8S component is composed of aggregates. If cytosol was preincubated with labeled androgen, followed by an incubation with unlabeled androgen, and subsequently analyzed for binding on low-ionic strength gradients, only the congruent to 8S peak could be detected, indicating that most of the binding in the congruent to 4.6S region was rapidly dissociable. This suggests that binding in this region was to moieties other than receptor. Since androgen binding proteins (ABP) of testicular origin would have been cleared from the epididymis at the timepoints that we concentrated on for most of these studies, the 4.6S binding probably represents the association of androgen with plasma testosterone binding globulin (TeBG). The binding of androgen to the receptor can be inhibited by cyproterone, while this antiandrogen does not inhibit binding to either ABP or TeBG at the concentration used.     

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.     

Comparison of male reproductive parameters in three rat strains: Dark Agouti, Sprague-Dawley and Wistar

The choice of experimental animal can have a large impact on experimental results, an example is the anecdotal evidence suggesting that Dark Agouti (DA) rats have a lower reproductive capacity than other rat strains. In this paper we report on an investigation into male reproductive characteristics in three rat strains--Wistar, Sprague-Dawley (outbred strains) and DA (an inbred strain). Reproductive organ weights, blood testosterone levels and sperm counts were measured in mature age-matched male rats. DA animals had significantly smaller testis weights than the Sprague-Dawley and Wistar animals, and this did not appear to be related to the overall smaller body mass of the DAs. There were no differences between the three strains in testicular histology or sperm counts (per gram testis). Although there was also no significant difference in epididymal sperm count, the DA animals had a much greater variability in sperm count than the other strains. There were no differences in relative (to body weight) epididymal, seminal vesicle or ventral prostate weights or in the blood testosterone levels. These results suggest that differences in reproductive capacity in DAs are neither the result of morphological differences in the reproductive organs nor in circulating testosterone levels. Sperm production appears to be normal but the lowered testicular weight and variability in epididymal sperm counts suggests that there are other factors in the testicular or epididymal environment which alter male reproductive function.     

Inhibition of prolactin secretion and androgenic function in the adult male rat

The effects of bromocriptine induced hypoprolactinemia on the testicular function were studied in adult rats. Bromocriptine treatment (1500 micrograms/day for 24 days) reduced serum and pituitary Prolactin levels, indicating a decrease in prolactin secretion and synthesis. No change in reproductive organ weights was seen in treated animals. Hypoprolactinemia had no effect on plasma testosterone or androstenedione levels and testicular androstenedione content, but decreased significantly testicular testosterone content. These findings indicated that experimental hypoprolactinemia induced a decrease in testicular testosterone content without affecting androgens levels.     

Serum concentrations of reproductive hormones after administration of various anesthetics to immature and young adult male rats

Immature and young adult male rats were either castrated or unoperated. One of seven anesthetic agents (Rompun, Bio-Tal, Thiopental, pentobarbital, ketamine, halothane, or ether) was administered. When the animals were clearly anesthetized, they were decapitated. Control rats were decapitated without anesthesia. Serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, testosterone, and androstenedione were determined by radioimmunoassay. None of the anesthetics was clearly suitable for study of all these hormones. Most would be suitable for acute LH studies. Ketamine and halothane appeared inappropriate for FSH studies in immature rats. Pentobarbital, Rompun, and ether caused increases in serum prolactin. Most of the agents appeared to cause a reduction in serum testosterone in intact rats but an increase in castrated animals, suggesting an inhibition of testicular androgen secretion and a stimulation of adrenal androgen secretion.     

Altered bioavailability of testosterone in androgen-binding protein-transgenic mice

Serum and intra-testicular total and free testosterone levels in different age groups of mice (7-360-day-old) were analyzed by radioimmunoassay (RIA) in age-matched wild type (WT)-control and in transgenic mice homozygous to rat androgen-binding protein (ABP-TG), in order to identify possible causes of increased pre-pubertal germ cell apoptosis, spermatogenetic defect and reduced fertility seen in ABP-TG mice. Total intra-testicular testosterone levels in the pre-pubertal ABP-TG (7, 14, 21 and 30-day-old) mice were significantly lower than those in age-matched WT-controls. After puberty (60 days and older) the total intra-testicular testosterone levels were higher than those in age-matched WT-controls and increased gradually, peaking on day 180. Serum total testosterone levels in ABP-TG mice did not differ from those in WT-control until day 30. However, a significant increase in the level of serum total testosterone was observed from day 60. Serum and intra-testicular free testosterone levels were significantly lower in 30, 120, 180 and 360-day-old ABP-TG mice than in age-matched WT-controls. Immunohistochemistry for the cholesterol side-chain cleavage (cytochrome P450) enzyme and quantitative real-time RT-PCR analysis of mRNAs for androgen receptor and for enzymes related to steroidogenesis did not show any changes in 30-day-old ABP-TG mice, indicating that the rates of steroidogenesis and utilization were not altered. Human chorionic gonadotrophin (hCG) administration to adult ABP-TG mice increased the intra-testicular total and free testosterone as well as total germ cell counts. We conclude that the presence of greater than physiological concentration of ABP in the mouse testis alters the ratio of free/bound testosterone, and thereby decreases the availability of free testosterone. As a result, a heightened wave of germ cell apoptosis during the pre-pubertal period followed by a reduction in germ cell numbers and reduced fertility is seen in these mice.     

Effect of long term diazepam administration on testicular benzodiazepine receptors and steroidogenesis.

We evaluated the effect of acute and chronic diazepam administration on testicular peripheral type benzodiazepine receptors (PBZD-R), serum testosterone and LH levels and the "in vitro" androgen production in response to Ro 5-4864, a PBZD-R agonist. The chronic diazepam treatment induced a significant fall in plasma testosterone concentration while LH levels remained unchanged. The number of PBZD-R was reduced by 37% and low concentrations (10(-8)-10(-6) M) of Ro 5-4864 failed to stimulate "in vitro" androgen production. The acute diazepam administration caused a significant increase in plasma testosterone levels while no changes were observed in LH concentrations and testicular PBZD-R. These results further suggest a modulatory role of PBZD-R on testicular steroidogenic activity.