Dihydrotestosterone Supplementation Research Articles

ZIP9 mediates the effects of DHT on learning, memory and hippocampal synaptic plasticity of male Tfm and APP/PS1 mice.

Androgens are closely associated with functions of hippocampal learning, memory, and synaptic plasticity. The zinc transporter ZIP9 (SLC39A9) regulates androgen effects as a binding site distinct from the androgen receptor (AR). However, it is still unclear whether androgens regulate their functions in hippocampus of mice through ZIP9. Compared with wild-type (WT) male mice, we found that AR-deficient male testicular feminization mutation (Tfm) mice with low androgen levels had learning and memory impairment, decreased expression of hippocampal synaptic proteins PSD95, drebrin, SYP, and dendritic spine density. Dihydrotestosterone (DHT) supplementation significantly improved these conditions in Tfm male mice, although the beneficial effects disappeared after hippocampal ZIP9 knockdown. To explore the underlying mechanism, we first detected the phosphorylation of ERK1/2 and eIF4E in the hippocampus and found that it was lower in Tfm male mice than in WT male mice, it upregulated with DHT supplementation, and it downregulated after hippocampal ZIP9 knockdown. Next, we found that the expression of PSD95, p-ERK1/2, and p-eIF4E increased in DHT-treated mouse hippocampal neuron HT22 cells, and ZIP9 knockdown or overexpression inhibited or further enhanced these effects. Using the ERK1/2 specific inhibitor SCH772984 and eIF4E specific inhibitor eFT508, we found that DHT activated ERK1/2 through ZIP9, resulting in eIF4E phosphorylation, thus promoting PSD95 protein expression in HT22 cells. Finally, we found that ZIP9 mediated the effects of DHT on the expression of synaptic proteins PSD95, drebrin, SYP, and dendritic spine density in the hippocampus of APP/PS1 mice through the ERK1/2-eIF4E pathway and affected learning and memory. This study demonstrated that androgen affected learning and memory in mice through ZIP9, providing new experimental evidence for improvement in learning and memory in Alzheimer's disease with androgen supplementation.

Impact of Ovariectomy on the Anterior Pituitary Gland in Female Rats.

Ovariectomy (OVX) causes a depletion of circulating estradiol (E2) and influences hypothalamic kisspeptin neurons, which govern gonadotropin-releasing hormone (GnRH) release and ultimately gonadotropin secretion. In this study, we examined the changes induced by OVX on the anterior pituitary gland in female rats. OVX significantly increased the mRNA expression of gonadotropin α, luteinizing hormone (LH) β, and follicle-stimulating hormone (FSH) β subunits within the pituitary gland compared with control (sham-operated) rats, and this was completely suppressed by E2 supplementation. High-dose dihydrotestosterone supplementation also prevented the OVX-induced increase in the expression of the three gonadotropin subunits. GnRH receptor mRNA expression within the pituitary was significantly increased in OVX rats, and this increase was completely inhibited by E2 supplementation. The mRNA expression of the receptors for adenylate cyclase-activating polypeptide and kisspeptin was unchanged by OVX. Although the mRNA levels of inhibin α, βA, and βB subunits within the pituitary gland were not modulated by OVX, follistatin gene expression within the pituitary gland was increased by OVX, and this increase was completely inhibited by E2 supplementation after OVX. In experiments using a pituitary gonadotroph cell model (LβT2 cells), follistatin itself did not modulate the mRNA expression of gonadotropin LHβ and FSHβ subunits, and the GnRH-induced increase in the expression of these genes was slightly inhibited in the presence of follistatin. Our current observations suggest that OVX induces several characteristic changes in the pituitary gland of rats.

From Conception to Adulthood: The Impact of Androgens on Abnormalities of Male Genital Development and Size

Androgens play a critical role in male development starting in utero. In a 46XY male patient, testosterone plays a significant role in the development of the internal genitalia while external genitalia formation is largely dependent on the conversion of testosterone to dihydrotestosterone (DHT). Testosterone is important for testicular descent and later, the onset of puberty. Defects in the production of testosterone, the conversion of testosterone to DHT, or within the androgen receptor can lead to a variety of pediatric urological conditions including micropenis, cryptorchidism, hypospadias, and disorders of sexual differentiation. Supplementation with testosterone is the standard of care for the treatment of micropenis and is used to increase glans size and reduce complications before hypospadias repair. Unfortunately, patients with a 5-alpha reductase deficiency will respond sub-optimally to testosterone supplementation and instead may benefit from DHT supplementation.

Fragile X Mental Retardation Protein Mediates the Effects of Androgen on Hippocampal PSD95 Expression and Dendritic Spines Density/Morphology and Autism-Like Behaviors Through miR-125a.

Dysregulated synaptic plasticity is a key feature of neurodevelopmental disorders, including autism. This study investigated whether Fragile X mental retardation protein (FMRP), a selective RNA-binding protein that regulates synaptic protein expression by interacting with miRNAs, mediates the effects of androgens that play an important role in regulating the synaptic plasticity in the hippocampus. Experiments using mouse hippocampal neuron HT22 cells demonstrated that dihydrotestosterone (DHT) increased the expression of postsynaptic density protein 95 (PSD95) by inhibiting FMRP expression. Administration of miR-125a inhibitor upregulated the PSD95 expression and significantly increased the DHT-induced upregulation of PSD95. FMRP knockdown in HT22 cells reduced the expression of miR-125a. Moreover, miR-125a inhibitor upregulated the PSD95 expression in the DHT-treated HT22 cells with FMRP knockdown. Subsequently, the effects of androgen-mediated via FMRP in regulating neural behaviors and PSD95 expression and dendritic spines density/morphology were investigated using Fmr1 knockout (KO) and wild-type littermate (WT) mice. The castration of WT mice reduced the androgen levels, aggravated anxiety and depression, and impaired learning and memory and sociability of mice. DHT supplementation post-castration reversed the alterations in density and maturity of dendritic spines of hippocampal neurons and behavioral disorders in WT mice; however, it did not reveal such effects in Fmr1 KO mice. Further, immunohistochemical staining and western blotting analyses after knocking down miR-125a revealed similar effects of castration and post-castration DHT supplementation on PSD95 protein expression. These findings clarified that FMRP mediated the effects of DHT through miR-125a in regulating the expression of hippocampal synaptic protein PSD95. This study provides evidence for the neuroprotective mechanism of androgen in PSD95 expression and dendritic spines density/morphology and suggests that treatment interventions with androgen could be helpful for the management of synaptic plasticity disorders.

Androgen action on renal calcium and phosphate handling: Effects of bisphosphonate treatment and low calcium diet

Renal calcium and phosphate handling is an important contributor to mineral homeostasis and bone health and the androgen receptor (AR) is highly expressed in the kidney. We investigated the short term effects of androgen deprivation on renal calcium and phosphate reabsorption, independent of their effects on bone. Two weeks following orchidectomy (ORX) of adult mice, bone loss occurred along with hypercalciuria, which was similarly prevented by testosterone and dihydrotestosterone supplementation. Treatment with bisphosphonates prior to ORX also inhibited hypercalciuria, indicating that the calcium flux originated from the bone. Renal calcium and phosphate transporter expression was increased post-ORX, independent of bisphosphonates. Furthermore, androgen deprivation appeared to stimulate local synthesis of 1,25(OH)2D3. When bisphosphonate-treated mice were fed a low calcium diet, bone resorption was no longer blocked and secondary hyperparathyroidism developed, which was more pronounced in ORX mice than sham-operated mice. In conclusion, this study shows that androgen deprivation increased renal calcium and phosphate transporter expression, independent of bone, and underlines the importance of adequate intestinal calcium supply in circumstances of androgen deprivation and bisphosphonate treatment.

Neuroprotection by dihydrotestosterone in LPS-induced neuroinflammation

Microglia-induced neuroinflammation plays a vital role in the etiology and progression of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis. The neuroprotective role of androgens, including testosterone and its metabolite dihydrotestosterone (DHT), has been increasingly demonstrated in these diseases, but few studies investigated the effects of androgen on neuroinflammation. This study investigated the role of DHT in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage and behavioral dysfunction, as well as underlying mechanisms. We showed that DHT inhibited LPS-induced release of proinflammatory factors, including TNF-α, IL-1β, IL-6; iNOS, COX-2, NO, and PGE2 in BV2 cells and primary microglia by suppressing the TLR4-mediated NF-κB and MAPK p38 signaling pathways, thus protecting SH-SY5Y neurons from inflammatory damage induced by activated microglia. In an LPS-induced neuroinflammation mouse model, endogenous DHT depletion by castration exacerbated inflammatory responses by upregulating the levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 in the serum and brain by increasing the LR4-mediated NF-κB and MAPK pathway activation, but these effects were restored by exogenous DHT supplementation. Moreover, DHT also regulated the mRNA levels of the anti-inflammatory cytokines IL-10 and IL-13 in the brain. In addition, DHT modulated the expression of Aβ, the apoptotic proteins caspase-3, Bcl-2, and Bax, and synaptophysin, as well as neuronal damage in LPS-treated mouse brains. Further behavioral tests revealed that DHT ameliorated LPS-induced spatial and learning impairment and motor incoordination, and partly improved the locomotor activity in LPS-injected mice. Therefore, this study suggests that DHT exerts anti-neuroinflammatory and neuroprotective effects; thus, androgen replacement therapy is a potential therapeutic strategy for improving cognitive and behavioral function in neuroinflammation-related diseases.

Androgen Deficiency Exacerbates High-Fat Diet-Induced Metabolic Alterations in Male Mice.

Androgen deficiency is associated with obesity, metabolic syndrome, and type 2 diabetes mellitus in men, but the mechanisms behind these associations remain unclear. In this study, we investigated the combined effects of androgen deficiency and high-fat diet (HFD) on body composition and glucose homeostasis in C57BL/6J male mice. Two models of androgen deficiency were used: orchidectomy (ORX) and androgen receptor knockout mice. Both models displayed higher adiposity and serum leptin levels upon HFD, whereas no differences were seen on a regular diet. Fat accumulation in HFD ORX animals was accompanied by increased sedentary behavior and occurred in spite of reduced food intake. HFD ORX mice showed white adipocyte hypertrophy, correlated with decreased mitochondrial content but not function as well as increased lipogenesis and decreased lipolysis suggested by the up-regulation of fatty acid synthase and the down-regulation of hormone-sensitive lipase. Both ORX and androgen receptor knockout exacerbated HFD-induced glucose intolerance by impairing insulin action in liver and skeletal muscle, as evidenced by the increased triglyceride and decreased glycogen content in these tissues. In addition, serum IL-1β levels were elevated, and pancreatic insulin secretion was impaired after ORX. Testosterone but not dihydrotestosterone supplementation restored the castration effects on body composition and glucose homeostasis. We conclude that sex steroid deficiency in combination with HFD exacerbates adiposity, insulin resistance, and β-cell failure in 2 preclinical male mouse models. Our findings stress the importance of a healthy diet in a clinical context of androgen deficiency and may have implications for the prevention of metabolic alterations in hypogonadal men.

Combined inhibition of aromatase activity and dihydrotestosterone supplementation attenuates renal injury in male streptozotocin (STZ)-induced diabetic rats.

Our previous studies showed that streptozotocin (STZ)-induced diabetic male rats have increased estradiol and decreased testosterone levels that correlate with renal injury (Xu Q, Wells CC, Garman GH, Asico L, Escano CS, Maric C. Hypertension 51: 1218-1224, 2008). We further showed that either supplementing dihydrotestosterone (DHT) or inhibiting estradiol biosynthesis in these diabetic rats was only partially renoprotective (Manigrasso MB, Sawyer RT, Marbury DC, Flynn ER, Maric C. Am J Physiol Renal Physiol 301: F634-F640, 2011; Xu Q, Prabhu A, Xu S, Manigrassso MB, Maric C. Am J Physiol 297: F307-F315, 2009). The aim of this study was to test the hypothesis that the combined therapy of DHT supplementation and inhibition of estradiol synthesis would afford better renoprotection than either treatment alone. The study was performed in 12-wk-old male nondiabetic (ND), STZ-induced diabetic (D), and STZ-induced diabetic rats that received the combined therapy of 0.75 mg/day of DHT along with 0.15 mg · kg(-1) · day(-1) of an aromatase inhibitor, anastrozole (Dta), for 12 wk. Treatment with the combined therapy resulted in attenuation of albuminuria by 84%, glomerulosclerosis by 55%, and tubulointerstitial fibrosis by 62%. In addition, the combined treatment decreased the density of renal cortical CD68-positive cells by 70% and decreased protein expression of transforming growth factor-β protein expression by 60%, collagen type IV by 65%, TNF-α by 55%, and IL-6 by 60%. We conclude that the combined treatment of DHT and blocking aromatase activity in diabetic male STZ-induced diabetic rats provides superior treatment than either treatment alone in the prevention of diabetic renal disease.

Regional Expression of Androgen Receptor Coregulators and Androgen Action in the Mouse Epididymis

Endocrine regulation of the mouse initial segment (IS) and distal caput epididymides was studied using genome-wide profiling of gene expression. Among the IS-enriched genes, 29% were significantly down-regulated 1 day after gonadectomy. Of those genes, dihydrotestosterone (DHT) supplementation was not sufficient to maintain their pregonadectomy level of expression in 70%. Of the caput-enriched genes, 16% were significantly down-regulated after gonadectomy, and of those genes, DHT supplementation did not maintain the initial level of expression in 28%. Identical data were obtained by clustering analyses performed for the expression data of epididymal genes. Furthermore, the microarray data revealed that 26 androgen receptor coregulators were expressed in the epididymis, of which several were confirmed by quantitative reverse transcriptase polymerase chain reaction analysis. This suggests putative involvement of these proteins in the segment-specific regulation of the epididymal genes. The pattern of epididymal gene expression in the novel proximal epididymis-specific androgen receptor knockout mouse ProxE-ARKO, with severe hypotrophy and hypoplasia of the caput epithelium, furthermore suggested that a subset of genes whose expression cannot be maintained by systemic androgen alone still require either direct lumicrine androgen action or a permissive effect of circulating testosterone. It is evident that testicular factors, one of which could be the high-concentration luminal androgen, are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by systemic androgens.

Dose-dependent effects of dihydrotestosterone in the streptozotocin-induced diabetic rat kidney

We recently reported that castration exacerbates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis associated with diabetic renal disease. The aim of the present study was to examine whether these effects of castration can be attenuated with dihydrotestosterone (DHT) supplementation. The study was performed in castrated male Sprague-Dawley, streptozotocin-induced diabetic rats treated with 0 mg/day DHT (DHT(0)), 0.75 mg/day DHT (DHT(0.75)), or 2.0 mg/day DHT (DHT(2.0)) for 14 wk. Treatment with 0.75 mg/day DHT attenuated castration-associated increases in urine albumin excretion (DHT(0), 81.2 +/- 18.1; DHT(0.75), 26.57 +/- 5.8 mg/day; P < 0.05), glomerulosclerosis (DHT(0), 1.1 +/- 0.79; DHT(0.75), 0.43 +/- 0.043 arbitrary units; P < 0.001), tubulointerstitial fibrosis (DHT(0), 1.3 +/- 0.12; DHT(0.75), 1.1 +/- 0.096 AU; P < 0.05), collagen type IV [DHT(0), 3.2 +/- 0.11; DHT(0.75), 2.1 +/- 0.070 relative optical density (ROD); P < 0.01], transforming growth factor-beta (DHT(0), 3.2 +/- 0.16; DHT(0.75), 2.1 +/- 0.060 ROD; P < 0.01), IL-6 (DHT(0), 0.37 +/- 0.011; DHT(0.75), 0.27 +/- 0.014 ROD; P < 0.05), and protein expression and reduced CD68-positive cell abundance (DHT(0), 17 +/- 0.86; DHT(0.75), 4.4 +/- 0.55 cells/mm(2); P < 0.001). In contrast, treatment with 2.0 mg/day DHT exacerbated all these parameters. These data suggest that the detrimental effects of castration in the diabetic kidney can be attenuated with low doses of DHT, whereas high doses augment the adverse effects of castration, and these effects appear to be influenced by estradiol. We conclude that the effects of DHT are dose dependent but caution should be taken when DHT supplementation is considered in the treatment of diabetic renal disease.

Hepatocellular Carcinoma Associated with Liver-Gender Disruption in Male Mice

Hepatocellular carcinoma (HCC) is a male-predominant cancer associated with chronic hepatitis. Like human viral hepatitis, murine Helicobacter hepaticus infection produces inflammation and HCC with a masculine bias. We used this model to identify potential mechanisms of male HCC predisposition. Male weanling A/JCr mice (n = 67) were gavaged with H. hepaticus or vehicle. At 1 year, mice were distributed into four groups: surgical castration, chemical castration, castration followed by dihydrotestosterone supplementation, or sexually intact controls. Responses to infection were compared with IFN-gamma challenge alone. At 21 months, there was no significant difference in hepatitis between groups. Neither castration nor androgen receptor agonism altered tumor incidence. Infected mice with severe, but not mild, disease exhibited a mosaic of alterations to sexually dimorphic genes and microsomal long-chain fatty acids. By microarray, tumorigenic hepatitis was strongly associated with liver-gender disruption, defined as the loss of a gender-identifying hepatic molecular signature. IFN-gamma alone produced similar changes, demonstrating a role for proinflammatory cytokines in this process. In conclusion, hepatocarcinogenesis in male mice with chronic hepatitis is maturationally imprinted and androgen-independent. Proinflammatory cytokines may promote HCC in a male-predominant fashion due to high sensitivity of the masculinized liver to loss of sex-specific transcriptional balance. Liver-gender disruption has pleiotropic implications for hepatic enzyme activity, lipid processing, nuclear receptor activation, apoptosis, and proliferation. We propose a multistep model linking chronic hepatitis to liver cancer through cytokine-mediated derangement of gender-specific cellular metabolism. This model introduces a novel mechanism of inflammation-associated carcinogenesis consistent with male-predominant HCC risk.

MORPHOLOGICAL STUDY OF THE SPINAL MOTONEURONS CONTROLLING THE URETHRAL SPHINCTER OF FEMALE RATS: ROLE OF ANDROGENS IN A MENOPAUSAL MODEL

In this study in a menopausal rat model we describe the effect of androgen withdrawal on motoneurons innervating 2 muscles involved in female urinary continence, namely the intrinsic urethral sphincter and the pubococcygeus muscle. The 5 groups studied were 2 ovariectomized groups and their sham operated controls plus 1 ovariectomized group with dihydrotestosterone supplementation. Blood testosterone assays were performed. Androgen receptor immunohistochemistry was performed on spinal cord sections, in which specific motoneuron subpopulations were identified. Maximal somal cross-sectional area was compared among the groups. Differences in somal cross-sectional area of motoneurons innervating the pubococcygeus muscle were observed after ovariectomy. Androgen receptor immunopositive nuclei staining disappeared, in line with undetectable blood testosterone levels. The modifications of androgen receptor expression and somal morphological appearance in the pudendal motoneurons controlling the external urethral sphincter of ovariectomized female rats suggests that testosterone probably influences neurotransmission and coordination of urinary continence at the spinal level.

Androgen dependency of hepatocarcinogenesis in TGFalpha transgenic mice.

Sex difference has been shown to play a major role in susceptibility to the development of hepatocellular carcinoma in humans and rodents. In order to clarify the necessity of androgens in hepatic tumorigenesis in transgenic mice overexpressing transforming growth factor (TGF) alpha (MT42), androgen supplement after castration and the LH-RH analogue, leuprolerin acetate, were tested in an experimental model in MT42. Male MT42 mice were castrated and supplemented with dihydrotestosterone (DHT) every three months up to 15 months and hepatic tumorigenesis was observed. Leuprolerin acetate was administered to both male and female MT42 mice once a month from 2 months after birth to 15 months to observe the effect on hepatic tumorigenesis. Northern hybridization was performed to detect messenger RNA (mRNA) of TGFalpha expression and the rate of proliferative cell nuclear antigen (PCNA) staining compared with the castrated and non-treated mice. Castration tended to decrease both body and liver weight in MT42 mice which was then restored by DHT. Untreated MT42 males developed 11 liver tumors in 6 mice. Hormonal treatment including castration and DHT supplementation did not change the expression of TGFalpha-mRNA. Castrated transgenic mice developed 2 liver tumors in 2 out of 6 mice and DHT supplementation after castration restored the number of liver tumors to 9 in 5 of 6 mice. PCNA labelling indexes of liver tumors and adjacent non-tumorous-liver were 7.1% (p<0.05): 0.6% in untreated MT42, 3.2%: 0.2% in castrated MT42 and 10.1% (p<0.05): 0.5% in MT42 with castration and DHT supplementation (significant difference compared with castrated mice). Leuprolerin acetate-treated MT42 males developed one liver tumor in 6 mice compared to MT42 administered with saline as a vehicle control in which group 7 liver tumors in 6 male MT42 were observed. Tumors in castrated-MT42 and leuprolerin treated-MT42 were smaller than those in control MT42 mice. TGFalpha related hepatocarcinogenesis and hepatocyte proliferation are increased by androgenic stimulation. Suppression of androgens may be useful for the treatment of TGFalpha related liver tumors.

Transdifferentiation of cultured human prostate cancer cells to a neuroendocrine cell phenotype in a hormone-depleted medium

Neuroendocrine (NE) cells are enigmatically found in association with human prostate cancers and their numbers are reported to increase in advanced and hormoneresistant tumors. The origin of this cell type and the reason for their appearance in prostate tumors remains unresolved. Previously, Bang et al. (Proc Natl Acad Sci USA 1994;91:5330) reported that dibutyryl adenosine 3′,5′-cyclic phosphate (db-cAMP), an agent that upregulates intracellular cAMP, was able to induce a NE cell-like phenotype of cultured human prostate cancer cells, including the androgen-sensitive LNCaP line. Here we report that chronic incubation of LNCaP cells in a medium containing 10% charcoal-stripped fetal bovine serum (CSFBS) likewise induces NE differentiation of these cells. Within 5 days of switching low density cultures of LNCaP cells to this modified medium, the cells growth arrest and acquire an altered morphology with numerous cytoplasmic secretory granules and elongated processes that resemble cultured neurons. This morphology predominates at 10 days with complete transformation seen by 20 days of culture. Electron microscopic analysis of sections of CS-FBS maintained cells showed the presence of abundant dense core secretory granules characteristic of NE cells. Immunohistochemical staining identified the upregulation of the expression of NE markers bombesin, neuron-specific enolase, and S-100 in this modified culture medium. Once established, the NE cell-like phenotype was found to be reversible upon replacement with a medium containing unmodified fetal bovine serum, but not by direct supplementation of CS-FBS medium with dihydrotestosterone (DHT) (I nM). DHT supplementation did, however, suppress the development of the NE cell-like phenotype when it was present at the initiation of exposure to CS-FBS medium. In contrast to db-cAMP treatment, which did not affect prostate specific antigen (PSA) or androgen receptor (AR) expression of LNCaP cells, NE-differentiated LNCaP cells derived in this hormone-deficient medium showed marked downregulation of PSA and AR expression. These in vitro results further support the concept that prostate cancer cells can tranform in vivo to cells with a NE phenotype and suggest that this transformation might be accelerated in patients by certain therapies for prostate cancer.

Persistence of infertility in GnRH immunized male rats treated with subdermal implants of dihydrotestosterone (DHT)

Male hormonal contraception has been limited to date because two fundamental requirements have not been concurrently satisfied, these are, consistent and dependable azoospermia and infertility coupled with maintenance of libido. The objective of this study was to determine the extent to which implants of potent androgen (DHT) will restore androgenization and spermatogenesis in hypogonadotropic infertile male rats. Twenty-five sexually mature male rats of proven fertility were actively immunized against gonadotropin releasing hormone (GnRH) to induce azoospermia. After azoospermia was achieved, GnRH immunized rats received subdermal DHT-filled Silastic implants of 2, 4, 6, or 8 cm, or empty implants (n=5/group). Five untreated control rats received empty capsules. Eight weeks later, fertility was evaluated, sperm number was obtained from the testis, and weights of androgen-dependent organs were measured. The results indicate that immunoneutralization of GnRH induced complete azoospermia, and subsequent treatment with DHT implants of 2 or 4 cm for 8 wk restored accessory organ weights, but did not restore spermatogenesis or fertility. In addition, DHT implants of 6 to 8 cm partially restored spermatogenesis, but not fertility. We conclude that low-dose DHT supplementation of GnRH-immunized rats may be a suitable alternate therapy able to maintain androgenization in the face of persistent azoospermia in the rat. This may be an effective model for development of a male contraceptive.