Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges.

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14-/- mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.

Curcumin treatment leads to better cognitive and mood function in a model of Gulf War Illness with enhanced neurogenesis, and alleviation of inflammation and mitochondrial dysfunction in the hippocampus.

Diminished cognitive and mood function are among the most conspicuous symptoms of Gulf War Illness (GWI). Our previous studies in a rat model of GWI have demonstrated that persistent cognitive and mood impairments are associated with substantially declined neurogenesis, chronic low-grade inflammation, increased oxidative stress and mitochondrial dysfunction in the hippocampus. We tested the efficacy of curcumin (CUR) to maintain better cognitive and mood function in a rat model of GWI because of its neurogenic, antiinflammatory, antioxidant, and memory and mood enhancing properties. Male rats were exposed daily to low doses of GWI-related chemicals, pyridostigmine bromide, N,N-diethyl-m-toluamide (DEET) and permethrin, and 5-minutes of restraint stress for 28 days. Animals were next randomly assigned to two groups, which received daily CUR or vehicle treatment for 30 days. Animals also received 5'-bromodeoxyuridine during the last seven days of treatment for analysis of neurogenesis. Behavioral studies through object location, novel object recognition and novelty suppressed feeding tests performed sixty days after treatment revealed better cognitive and mood function in CUR treated GWI rats. These rats also displayed enhanced neurogenesis and diminished inflammation typified by reduced astrocyte hypertrophy and activated microglia in the hippocampus. Additional studies showed that CUR treatment to GWI rats enhanced the expression of antioxidant genes and normalized the expression of multiple genes related to mitochondrial respiration. Thus, CUR therapy is efficacious for maintaining better memory and mood function in a model of GWI. Enhanced neurogenesis, restrained inflammation and oxidative stress with normalized mitochondrial respiration may underlie better memory and mood function mediated by CUR treatment.

Rapid implementation of triaging system for assessment of breast referrals from primary care centres during the COVID-19 pandemic.

OBJECTIVE: The aim of this study was to establish a triaging system for assessment of breast referrals from primary care to ensure safe and effective breast services without compromising breast cancer management. BACKGROUND: COVID-19 was officially declared a global pandemic on 11 March 2020, and with no effective treatment available, preventing spread has been paramount. Previously, all referrals from primary care were seen in the rapid-access breast clinic (RABC). Clinic appointments exposed patients and healthcare professionals to risk. METHOD: Initial triage during the lockdown was in line with national governing body guidance, rejected low risk referrals and streamed remaining patients through a telephone consultation to RABC or discharge. A modified triage pathway streamed all patients through virtual triage to RABC, telephone clinic or discharge with advice and guidance categories. Demographics, reasons for referral and outcomes data were collected and presented as median with range and frequency with percentages. RESULTS: Initial triage (23 March-23 April 2020) found fewer referrals with a higher percentage of breast cancer diagnoses. Modified triage (22 June-17 July 2020) resulted in a 35.1% (99/282) reduction in RABC attendance. Overall cancer detection rate remained similar at 4.2% of all referrals pre-COVID (18/429) and 4.3% (12/282) during modified triage. After six months follow-up of patients not seen in RABC during the modified triage pathway, 18 patients were re-referred to RABC and none were diagnosed with cancer. CONCLUSION: A modified triage pathway has the potential to improve triage efficiency and prevent unnecessary visits during the COVID-19 pandemic. Further refinement of pathway is feasible in collaboration with primary care.

Cost and cost-effectiveness of health behavior change interventions implemented with self-help groups in Bihar, India.

INTRODUCTION: Health interventions implemented with self-help groups (SHGs) enhance the relevance and acceptability of the health services. The Parivartan program was implemented in eight districts of Bihar with women's self-help groups to increase adoption of maternal and newborn health behaviors through layering health behavior change communication. This study estimates the cost and cost-effectiveness of a health behavior change program with SHGs in Bihar. METHODS: Cost analysis was conducted from a provider's perspective. All costs have been presented in US dollars for the purpose of international comparisons and converted to constant values. The effectiveness estimate was based on the reported changes in select newborn care practices. A decision model approach was used to estimate the potential number of neonatal deaths averted based on adoption of key newborn care practices. Using India's life expectancy of 65 years, cost per life year saved was calculated. A one-way sensitivity analysis was conducted using the upper and lower estimates for various variables in the model, and functionality of SHGs. RESULTS: The cost of forming an SHG group was US$254 and that of reaching a woman within the group was US$19. The unit cost for delivering health interventions through the Parivartan program was US$148 per group and US$11 per woman reached. During an 18 months period, Parivartan program reached around 17,120 SHGs and an estimated 20,544 pregnant women resulting in an estimated prevention of 23 neonatal deaths at a cost of US$3,825 per life year saved. CONCLUSION: SHGs can be an effective platform to increase uptake of women's health interventions and follow-up care, and also to broaden their utility beyond microfinance, particularly when they operate at a larger scale.

Effect of hormone modulations on donor-derived spermatogenesis or colonization after syngeneic and xenotransplantation in mice.

BACKGROUND: Cytotoxic cancer treatments, such as irradiation, can cause permanent sterility in male mammals owing to the loss of spermatogonial stem cells. In animal models, spermatogenesis could be restored from transplanted spermatogonial stem cells. Previously, we showed that transient suppression of FSH, LH, and testosterone in the recipient with a gonadotropin-releasing hormone antagonist (GnRH-ant), given immediately after irradiation, enhanced spermatogenesis from transplanted spermatogonial stem cells in mice and monkeys. OBJECTIVES: To explore improvements in the preparation of the recipient for efficient and reliable spermatogenic recovery from spermatogonial stem cell transplantation, so that it can be used effectively in clinical practice. MATERIALS AND METHODS: In mouse recipients, we evaluated the effects of hormone suppression given after germ cell depletion was complete, which is a more clinically relevant model, and also the importance of total androgen ablation and maintenance of FSH levels. Three regimens, GnRH-ant, GnRH-ant plus flutamide (androgen receptor antagonist), and GnRH-ant plus FSH, were administered prior to and around the time of transplantation of testis cells from immature mice or from prepubertal monkeys. RESULTS: Treatment with GnRH-ant resulted in a fourfold increase in spermatogenic recovery from GFP-marked transplanted mouse cells. Total androgen ablation with the addition of flutamide, started two weeks before transplantation, did not further enhance recovery. Surprisingly, FSH supplementation, started around the time of transplantation, actually reduced spermatogenic recovery from transplanted spermatogonial stem cells in GnRH-ant-treated mice. When prepubertal monkey testicular cells were transplanted into nude mice that were given the same hormone treatments, the numbers of donor-derived colonies were independent of hormone treatment. DISCUSSION AND CONCLUSION: The enhancements in spermatogenic recovery may only occur when syngeneic or closely related donor-recipient pairs are used. These results are useful in further investigations in choosing a hormone suppression regimen in combination with spermatogonial transplantation as a treatment to restore fertility in primates after cytotoxic therapy.

Distal pancreatectomy with autologous islet transplantation in chronic pancreatitis patients: First case series from India.

Pancreatectomy and autologous islet transplantation (AIT) are performed in highly specialized centers to alleviate abdominal pain and preserve endocrine function in patients with chronic pancreatitis. We aimed at initiating AIT studies in India for the first time in patients undergoing distal pancreatectomy (DP) to prevent development of diabetes. Four out of 14 chronic pancreatitis patients screened underwent DP with AIT. Pancreatectomy specimen preserved in Wisconsin solution was subjected to islet isolation employing standard protocol using collagenase V. Isolated islets were infused into the liver through portal vein after quality assessment and the four patients were followed for 1 year. During the study period, blood glucose, fasting insulin, and C-peptide were analyzed and glucose tolerance was assessed. Three patients could be infused with islets (4363 Islet equivalents, IEQ/kg, 3860 IEQ/kg, 3600 IEQ/kg) into liver without any infusion-related complications. Two of these patients maintained glucose tolerance and glycemic control (HbA1c: 6.0%) and one became diabetic at the end of 1 year (HbA1c: 7.5%). Circulating fasting insulin increased (2.7-24.4 µU/mL and 4.0-21.2 µU/mL) and C-peptide levels increased (2.2 to 3.6, 3.4-5.6 ng/mL) in these two patients. Circulating insulin was 2.7 µU/mL and C-peptide was 2.4 ng/mL in the patient who became diabetic at the end of 1 year, while insulin was 2.3 µU/mL and C-peptide was 1.2 ng/mL in the patient who could not be infused with islets after DP. Safety and feasibility of autologous islet transplantation is established in India for the first time.

Pedicle subtraction osteotomy for rigid post-tuberculous kyphosis.

We studied 15 patients with healed tuberculosis of the spine and a resultant kyphosis. We selected only those with no neurological deficit and performed a wedge resection of the vertebra using a transpedicular approach. The wedge was removed from the apex of the deformity. For those with a neurological deficit, we chose the conventional anterior debridement and decompression with 360 degrees circumferential fusion. At a mean follow-up of 26.8 months (8 to 46) the outcome was good with an increase in the mean Oswestry Disability Index from 56.26 (48 to 62) pre-operatively to 11.2 (6 to 16) at the latest follow-up.

Endoscopic screening and surveillance for Barrett's esophagus--clinical implications.

There is now a clear causal relationship between symptomatic gastroesophageal reflux and esophageal adenocarcinoma (Lagergren et al, 1999). The risk factor is now identified as Barrett's metaplasia (Solaymani et al, 2004). Chronic reflux results in Barrett's metaplastic change, and the route to carcinoma is a stepwise progression, through dysplasia to invasive carcinoma (Jankowski et al, 2000). Earlier-stage disease is found in patients undergoing surveillance and is the major predictor of survival following surgery (Fountoulakis et al, 2004). Screening and surveillance by endoscopic biopsy regimen has profound implications for the allocation of healthcare resources and the provision of clinical services. Screening a high-risk group such as men with gastroesophageal reflux disease (GERD) will result in the detection of more patients with Barrett's esophagus, many of whom are asymptomatic. Once detected, questions remain as to surveillance intervals and the current methodology for surveillance. There are profound challenges with the accurate endoscopic and pathologic detection and categorization of Barrett's metaplasia, dysplasia , and, indeed, cancer. New endoscopic detection methods are being investigated to improve the diagnosis and definition of the premalignant phenotype. The detection of dysplasia requires increased surveillance and usually intervention either endoscopically or with surgery.

Endoscopic screening and surveillance for Barrett's esophagus--clinical implications.

There is now a clear causal relationship between symptomatic gastroesophageal reflux and esophageal adenocarcinoma (Lagergren et al, 1999). The risk factor is now identified as Barrett's metaplasia (Solaymani et al, 2004). Chronic reflux results in Barrett's metaplastic change, and the route to carcinoma is a stepwise progression, through dysplasia to invasive carcinoma (Jankowski et al, 2000). Earlier-stage disease is found in patients undergoing surveillance and is the major predictor of survival following surgery (Fountoulakis et al, 2004). Screening and surveillance by endoscopic biopsy regimen has profound implications for the allocation of healthcare resources and the provision of clinical services. Screening a high-risk group such as men with gastroesophageal reflux disease (GERD) will result in the detection of more patients with Barrett's esophagus, many of whom are asymptomatic. Once detected, questions remain as to surveillance intervals and the current methodology for surveillance. There are profound challenges with the accurate endoscopic and pathologic detection and categorization of Barrett's metaplasia, dysplasia , and, indeed, cancer. New endoscopic detection methods are being investigated to improve the diagnosis and definition of the premalignant phenotype. The detection of dysplasia requires increased surveillance and usually intervention either endoscopically or with surgery.

Correcting deformity in total knee arthroplasty: Techniques to avoid the release of collateral ligaments in severely deformed knees.

Collateral ligament release is advocated in total knee arthroplasty (TKA) to deal with significant coronal plane deformities, but is also associated with significant disadvantages. We describe steps to avoid release of the collateral (superficial medial and lateral collateral) ligaments during TKA in severely deformed knees, while correcting deformity and balancing the knee.

Distinct downstream targets manifest p53-dependent pathologies in mice.

Mdm2, the principal negative regulator of p53, is critical for survival, a fact clearly demonstrated by the p53-dependent death of germline or conditional mice following deletion of Mdm2. On the other hand, Mdm2 hypomorphic (Mdm2Puro/Δ7-12) or heterozygous (Mdm2+/-) mice that express either 30 or 50% of normal Mdm2 levels, respectively, are viable but present distinct phenotypes because of increased p53 activity. Mdm2 levels are also transcriptionally regulated by p53. We evaluated the significance of this reciprocal relationship in a new hypomorphic mouse model inheriting an aberrant Mdm2 allele with insertion of the neomycin cassette and deletion of 184-bp sequence in intron 3. These mice also carry mutations in the Mdm2 P2-promoter and thus express suboptimal levels of Mdm2 entirely encoded from the P1-promoter. Resulting mice exhibit abnormalities in skin pigmentation and reproductive tissue architecture, and are subfertile. Notably, all these phenotypes are rescued on a p53-null background. Furthermore, these phenotypes depend on distinct p53 downstream activities as genetic ablation of the pro-apoptotic gene Puma reverts the reproductive abnormalities but not skin hyperpigmentation, whereas deletion of cell cycle arrest gene p21 does not rescue either phenotype. Moreover, p53-mediated upregulation of Kitl influences skin pigmentation. Altogether, these data emphasize tissue-specific p53 activities that regulate cell fate.

Leydig cells contribute to the inhibition of spermatogonial differentiation after irradiation of the rat.

Irradiation with 6 Gy produces a complete block of spermatogonial differentiation in LBNF1 rats that would be permanent without treatment. Subsequent suppression of gonadotropins and testosterone (T) restores differentiation to the spermatocyte stage; however, this process requires 6 weeks. We evaluated the role of Leydig cells (LCs) in maintenance of the block in spermatogonial differentiation after exposure to radiation by specifically eliminating functional LCs with ethane dimethane sulfonate (EDS). EDS (but not another alkylating agent), given at 10 weeks after irradiation, induced spermatogonial differentiation in 24% of seminiferous tubules 2 weeks later. However, differentiation became blocked again at 4 weeks as LCs recovered. When EDS was followed by treatment with GnRH antagonist and flutamide, sustained spermatogonial differentiation was induced in >70% of tubules within 2 weeks. When EDS was followed by GnRH antagonist plus exogenous T, which also inhibits LC recovery but restores follicle stimulating hormone (FSH) levels, the spermatogonial differentiation was again rapid but transient. These results confirm that the factors that block spermatogonial differentiation are indirectly regulated by T, and probably FSH, and that adult and possibly immature LCs contribute to the production of such inhibitory factors. We tested whether insulin-like 3 (INSL3), a LC-produced protein whose expression correlated with the block in spermatogonial differentiation, was indeed responsible for the block by injecting synthetic INSL3 into the testes and knocking down its expression in vivo with siRNA. Neither treatment had any effect on spermatogonial differentiation. The Leydig cell products that contribute to the inhibition of spermatogonial differentiation in irradiated rats remain to be elucidated.

Nitric oxide synthase gene therapy rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arteries of cholesterol-fed rabbits.

BACKGROUND: Hypercholesterolemia reduces nitric oxide bioavailability, manifested by reduced endothelium-dependent vascular relaxation, and also induces vascular adhesion molecule expression and inflammatory cell infiltration. We have previously shown that gene therapy with NO synthase in hypercholesterolemic rabbits substantially reverses the deficit in vascular relaxation. In the present study, we show that NO synthase gene therapy rapidly and substantially reduces vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration. METHODS AND RESULTS: Thirty male New Zealand White rabbits were maintained on a 1% cholesterol diet for 11 to 13 weeks, then underwent carotid artery gene transfer with Ad.nNOS or Ad.betaGal (recombinant adenoviruses expressing neuronal NO synthase or beta-galactosidase, respectively), or received medium alone in a sham procedure. Arteries were harvested at 1 and 3 days after gene transfer, and the following parameters were determined by immunohistochemical and image-analysis techniques: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, lipid deposition by oil red O staining, lymphocyte infiltration (CD43-positive cells), and monocyte infiltration (RAM-11-positive cells). In Ad.nNOS-treated arteries, all markers were significantly decreased relative to Ad. betaGal or sham-treated arteries within 3 days after gene transfer. Ad.nNOS had a particularly striking impact on monocyte infiltration; as early as 24 hours after gene transfer, Ad.nNOS-treated arteries had >3-fold fewer monocytes than Ad.betaGal- or sham-treated arteries. CONCLUSIONS: NO synthase gene therapy rapidly ameliorates several markers of atherosclerosis in the cholesterol-fed rabbit.

Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells.

OBJECTIVES: Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells. METHODS: We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence. RESULTS: Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells. CONCLUSIONS: Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.

Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.

OBJECTIVES: Gene therapy may provide new approaches to reduce vein graft failure following coronary or peripheral bypass surgery. The aim of this study was to investigate the relative efficacy of intraoperative adenoviral gene transfer to vein grafts, comparing transgene expression in vein grafts with that in matched native vessels in the same animal. In addition, we assessed the impact of bypass grafting on the cellular targets of gene transfer. METHODS: New Zealand White rabbits underwent interposition bypass grafting of the carotid artery, using the ipsilateral external jugular vein, which was infected with an adenovirus expressing beta-galactosidase immediately prior to bypass grafting (n = 16). The contralateral native jugular vein (n = 16) and carotid artery (n = 8) were infected concurrently with the same adenoviral preparation. After 3, 7 or 14 days, beta-galactosidase protein expression was quantified by ELISA, and specific cell types expressing beta-galactosidase were identified by X-Gal staining and by immunohistochemistry. RESULTS: After 3 days, endothelial cells were efficiently transduced in all vessels; medial smooth muscle cells were transduced infrequently. In contrast to jugular veins after gene transfer, endothelium in vein grafts showed expression of VCAM-1 and ICAM-1, and intense inflammation with CD18+ leukocytes. Transgene expression in vein grafts at day 3 was maintained at levels approximately 50% of that in ungrafted jugular veins, but continued to decrease through day 7. CONCLUSIONS: Although vascular injury in early venous bypass grafts reduces gene transfer efficacy, significant transgene expression is maintained for at least 7 days. These findings have important implications for intraoperative gene transfer strategies in vein grafts.

Increasing testicular temperature by exposure to elevated ambient temperatures restores spermatogenesis in adult Utp14b (jsd) mutant (jsd) mice.

Because mutations in the human UTP14C gene are associated with male infertility, we sought to develop a method for fertility restoration in azoospermic mice with a mutation in the orthologous Utp14b(jsd) (jsd) gene that have spermatogonial arrest. The method is based on our observation that elevation of testicular temperatures restores spermatogonial differentiation in jsd mutant mice. To non-surgically raise intrascrotal temperatures we placed these mice in incubators at different elevated ambient temperatures. Exposure of jsd/jsd mice to ambient temperatures of 34.5 °C or 35.5 °C for 24 days increased the proportion of tubules with spermatocytes from 0% in untreated controls to over 80%. As those higher temperatures interfere with spermatid differentiation, the mice were then transferred to incubators at 32-32.5 °C for the next 24 days. These environments allowed differentiation to progress, resulting in up to 42% of tubules having late spermatids and about half of the mutant mice having spermatozoa in testicular suspensions. When these spermatozoa were used in intracytoplasmic sperm injection, all gave rise to viable healthy offspring with normal weight gain and fertility. The successful restoration of fertility in Utp14b mutant mice suggests that transient testicular warming might also be useful for spermatogenesis recovery in infertile men with UTP14C gene mutations.

Gonadotropin-releasing hormone analogs stimulate and testosterone inhibits the recovery of spermatogenesis in irradiated rats.

We investigated the effects of GnRH analogs, different doses of testosterone (T), an androgen receptor antagonist (flutamide), and combinations of these on the recovery of spermatogenesis after irradiation. Treatment with a GnRH agonist (Lupron) for 10 weeks after irradiation reduced the intratesticular T concentration (ITT) to 4% of that in irradiated rats and serum FSH to undetectable levels without altering serum LH levels. Injection of a GnRH antagonist (Cetrorelix) at 3 weeks after irradiation suppressed LH, FSH, and ITT to <7%, 32%, and 10%, respectively, of levels in irradiated-only rats within 2 weeks; suppression was maintained for approximately 3 to 4 weeks. The percentage of tubules with differentiated germ cells (repopulation index, RI) was <0.6% at weeks 10 to 20 after irradiation. Spermatogenic recovery was induced by both the GnRH agonist (RI = 58% at week 10; 91% at week 20) and antagonist (RI = 70% at week 13). There was a dose-dependent suppression of testicular germ cell repopulation when T was combined with GnRH analogs. The ability of T to abolish the spermatogenic stimulatory effect of the GnRH antagonist was evident by the similar RI obtained for irradiated rats given antagonist + T or T alone. This suppression of GnRH-induced recovery of spermatogenesis by T could be reversed by flutamide. The RI best correlated with the degree of ITT suppression. In ITT-suppressed rats, the RI also showed an inverse correlation with serum T levels. Thus, T and/or its androgenic metabolites either directly or indirectly inhibit spermatogenic recovery after irradiation through an androgen receptor-mediated process. In addition, there was a close negative correlation between RI and FSH levels, and hence, a spermatogenic inhibitory role for FSH in the irradiated rats cannot be ruled out.

Stimulation of spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone antagonist treatment.

Male juvenile spermatogonial depletion (jsd) mutant mice are sterile because of spermatogenic failure and so may provide a model for genetically caused human male infertility. To test the effects of testosterone suppression therapy on spermatogenesis in jsd/jsd mice, we treated them with Nal-Glu, a GnRH antagonist. Treatment with Nal-Glu at 2500 microg/kg/day was started at 5.5 or 8 weeks of age and continued for 4 or 8 weeks. Differentiation of spermatogonia was evaluated by the percentage of tubules containing two or more spermatocytes (% of differentiating tubules). Nal-Glu treatment caused a marked decrease in the weights of the testes and seminal vesicles and intratesticular testosterone concentrations. However, in contrast to a value of 1.3% in untreated jsd/jsd mice, the mean % of differentiating tubules was 59.9% and 25.1% in treatment groups started at 5.5 and 8 weeks of age, respectively. We propose that spermatogonial differentiation in jsd/jsd mutant mice is sensitive to the high intratesticular levels of testosterone and can only proceed when testosterone production is suppressed.

Testosterone inhibits spermatogonial differentiation in juvenile spermatogonial depletion mice.

The juvenile spermatogonial depletion (jsd) mutation results in spermatogonial arrest after the first wave of spermatogenesis. In homozygous jsd mice in a hybrid background (C3HxB6) that were identified with microsatellite markers, the percentage of tubules showing differentiating germ cells [tubule differentiation index (TDI)] rapidly decreased after 7 weeks of age with a correlative increase in the intratesticular testosterone (ITT) levels. Treatment with a GnRH antagonist, Cetrorelix, suppressed ITT and stimulated spermatogonial differentiation at the end of treatment. When treated mice were killed 5-13.3 weeks after the end of treatment, the ITT progressively increased, and the TDI progressively declined, but there was a transient appearance of tubules with mature spermatids. To delineate the role of testosterone (T) in spermatogonial arrest, we gave 7.6-week-old jsd mice exogenous T and/or the androgen receptor antagonist flutamide with or without GnRH antagonist for 4 weeks. Flutamide alone moderately stimulated spermatogonial differentiation (TDI = 30%). GnRH antagonist increased the TDI to 73%, and the addition of flutamide to the GnRH antagonist treatment further increased it to 95%. When T was combined with GnRH antagonist treatment, ITT was increased, and the TDI was reduced to 7%. Addition of flutamide to this combination reversed the T inhibition of GnRH antagonist stimulation of spermatogonial differentiation to a TDI of 57%. ITT levels showed a good negative correlation to the TDI obtained with various treatments, but no such correlation was observed for FSH or LH levels. The results indicate that T inhibits the ability of spermatogonia to differentiate in jsd mice through an androgen receptor-mediated process.