The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

The National Physicians Cooperative: transforming fertility management in the cancer setting and beyond.

Once unimaginable, fertility management is now a nationally established part of cancer care in institutions, from academic centers to community hospitals to private practices. Over the last two decades, advances in medicine and reproductive science have made it possible for men, women and children to be connected with an oncofertility specialist or offered fertility preservation soon after a cancer diagnosis. The Oncofertility Consortium's National Physicians Cooperative is a large-scale effort to engage physicians across disciplines - oncology, urology, obstetrics and gynecology, reproductive endocrinology, and behavioral health - in clinical and research activities to enable significant progress in providing fertility preservation options to children and adults. Here, we review the structure and function of the National Physicians Cooperative and identify next steps.

Predictors of sperm recovery after cryopreservation in testicular cancer.

Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT) who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC) in men with TGCT. Men with NSGCT were more likely to be younger (P < 0.01) and had high cancer stage (II or III, P < 0.01) compared with men with seminoma. In our multiple regression model, NSGCT histology, use of density gradient purification, and fresh TMC > median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III), rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials) before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.

Melphalan, alone or conjugated to an FSH-ß peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo.

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the ß-chain of FSHß. We hypothesized that conjugating melphalan to FSHß peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHß peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHß consisting of amino acids 33 to 53 (FSHß (33-53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHß (33-53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHß (33-53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHß (33-53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHß (33-53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHß (33-53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.

Levels of the retinoic acid synthesizing enzyme aldehyde dehydrogenase-1A2 are lower in testicular tissue from men with infertility.

OBJECTIVE: To determine whether decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis. DESIGN: Observational analysis of testicular tissue samples, sperm indices, and serum hormone concentrations. SETTING: Two infertility centers in Chile. PATIENT(S): 32 infertile men and 11 control men. INTERVENTION(S): Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2, and 1A3, in testicular tissue by a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) peptide assay. MAIN OUTCOME MEASURE(S): ALDH isozyme levels compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, and serum and intratesticular hormone concentrations. RESULT(S): Men with infertility had statistically significantly reduced levels of ALDH1A2 but not ALDH1A1 or ALDH1A3 in their testicular tissue compared with men with normal spermatogenesis. The ALDH1A2 protein levels were strongly correlated with the number of germ cells found via testicular biopsy. CONCLUSION(S): These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine whether men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis.

Optimizing fertility preservation for pre- and postpubertal males with cancer.

Advances in diagnostic techniques and therapies have drastically improved cancer survival rates. However, the testes are highly susceptible to chemotherapy and radiation therapy, and spermatogenesis and semen parameters can be negatively impacted either permanently or transiently. Fertility preservation is an important issue to patients, and sperm banking has a positive psychological effect that helps patients cope emotionally with cancer. Innovations in sperm freezing and assisted reproductive technologies are improving the fecundity of males regardless of sperm parameters, and now even allow for fertility potential after gonadotoxic therapy. Experimental options also exist for prepubertal males, and hold promise for use in the future. At present, sperm cryopreservation is underutilized in the setting of a cancer diagnosis, and pretreatment referrals to fertility specialists are inconsistently offered. A multidisciplinary team approach is critical to educate families on the available options and help accomplish fertility preservation in a timely manner.

Raw and test-thaw semen parameters after cryopreservation among men with newly diagnosed cancer.

OBJECTIVE: To characterize sperm parameters from thawed semen samples of men with different cancers who cryopreserved semen before oncologic therapy. DESIGN: Retrospective cohort study. SETTING: Tertiary academic medical center. PATIENT(S): 1,010 semen samples collected between 1994 to 2010. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mean total motile count (TMC), change in percentage motility and percentage survival (100 * [postthaw % motility/raw % motility]) for each cancer compared with data from samples of men without cancer (the "procreative management" group), and proportion of postthaw samples with TMC >5 × 10(6). RESULT(S): The procreative management group had the best raw and postthaw semen quality. The best raw and postthaw semen quality for cancer patients occurred in those with prostate cancer (TMC of 155.1 and 53.2 × 10(6), respectively) and the worst in those with leukemias. Lymphoid leukemias demonstrated the worst raw TMC (26.8 × 10(6)), but myeloid leukemias displayed the worst postthaw TMC (6.9 × 10(6)). The testicular cancer group was the only group with a statistically significantly lower chance of having TMC >5 × 10(6). CONCLUSION(S): Men with testicular cancer were most commonly referred for sperm cryopreservation and were the only group that was statistically significantly less likely to have TMC >5 × 10(6) on postthaw semen analysis. The most severe reduction in TMC was seen in the myeloid leukemia group, suggesting that these patients along with men with testis cancer and those with lymphoid leukemia should be counseled to provide increased numbers of specimens for fertility preservation.

Δ9-Tetrahydrocannabinol (Δ9-THC) attenuates mouse sperm motility and male fecundity.

BACKGROUND AND PURPOSE: Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB(1) cannabinoid receptor agonist, and Δ(9) -tetrahydracannabinol (Δ(9) -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of Δ(9) -THC on male fecundity were determined. EXPERIMENTAL APPROACH: Effects of Δ(9) -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of Δ(9) -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by Δ(9) -THC-treated males. KEY RESULTS Pretreatment of sperm for 15 min with 1 µM Δ(9) -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 µM WIN 55,212-2 or 10 µM Δ(9) -THC for 30 min reduced sperm ATP levels. In sperm lacking CB(1) receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of Δ(9) -THC persisted. Administration of 50 mg·kg(-1) Δ(9) -THC to male mice just before mating caused a 20% decrease in embryonic litter size. CONCLUSIONS AND IMPLICATIONS: Δ(9) -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or Δ(9) -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB(1) receptor-dependent whereas that of Δ(9) -THC is not. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Identification of antigen-specific IgG in sera from patients with chronic prostatitis.

Antigen-specific vaccines are one of several molecularly targeted approaches under investigation as possible treatments for prostate cancer. Important to the development of vaccines is the identification of appropriate target antigens. We hypothesized that antigens of the prostate might be identified in patients with the chronic prostatitis/pelvic pain syndrome, a syndrome for which an autoimmune pathology has been proposed. Such antigens might represent naturally recognized target antigens of the prostate that could be investigated in the future as prostate tumor antigens. In this report, we used SEREX to identify proteins expressed in a prostate cDNA expression library recognized by IgG from the sera of patients with chronic prostatitis. Candidate proteins were evaluated using a panel of sera from 62 subjects with symptomatic prostatitis and 71 control male blood donors. We identified one protein that was recognized primarily in sera from subjects with prostatitis compared with controls. MAD-PRO-34, a nucleolar autoantigen, was recognized in 6/62 subjects and 0/71 controls (p = 0.00897). This protein had previously been identified as an autoantigen in patients with prostate cancer. In addition, the NY-CO-7 protein was recognized in 9/62 subjects and 3/71 controls (p = 0.0654). Two subjects had IgG specific for both the MAD-PRO-34 and NY-CO-7 gene products. Our results demonstrate that some patients with the chronic prostatitis/pelvic pain syndrome have autoantibodies to specific proteins. Proteins identified, and MAD-PRO-34 in particular, could be further investigated as potential prostate tumor antigens.

Effects of age on DNA double-strand breaks and apoptosis in human sperm.

OBJECTIVE: This study was designed to explore the relationship between men's age and DNA damage and apoptosis in human spermatozoa. DESIGN: Semen samples were collected from men between the ages of 20 and 57 years. Sperm DNA double-strand breaks were assessed using the neutral microgel electrophoresis (comet) assay, and apoptosis was estimated using the DNA diffusion assay. SETTING: Academic medical center. PATIENT(S): Sixty-six men aged 20 to 57 years were recruited from infertility laboratory and general populations and consented to donate a semen sample. Recruitment was determined by time and day of analysis; the only exclusions were for azoospermia, prostatitis, or prior cancer therapy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA damage and apoptosis in human sperm. RESULT(S): Age correlated with an increasing percentage of sperm with highly damaged DNA (range: 0-83%) and tended to inversely correlate with percentage of apoptotic sperm (range: 0.3%-23%). For example, percentage of sperm with highly damaged DNA, comet extent, DNA break number, and other comet measures was statistically significantly higher in men aged 36-57 years than in those aged 20-35 years, but percentage apoptosis was statistically significantly lower in the older group. Semen analysis showed percentage motility to be significantly higher in younger age groups. CONCLUSION(S): This study clearly demonstrates an increase in sperm double-stranded DNA breaks with age. Our findings also suggest for the first time an age-related decrease in human sperm apoptosis. These novel findings may indicate deterioration of healthy sperm cell selection process with age.

Prostate biopsy culture findings of men with chronic pelvic pain syndrome do not differ from those of healthy controls.

PURPOSE: Previous reports have identified bacteria in the prostate of men with chronic pelvic pain syndrome. To examine whether prostatic bacteria are more prevalent among patients with chronic pelvic pain syndrome than among those without pelvic pain, we compared 4-glass urine test and prostate biopsy results. MATERIALS AND METHODS: A total of 120 patients with types IIIa and IIIb chronic pelvic pain syndrome and 60 asymptomatic controls underwent a standard 4-glass urine test, examination of expressed prostatic secretion leukocytes by hemocytometer and transperineal, digitally guided prostate biopsies. Tissue was cultured for aerobes, anaerobes, Trichomonas vaginalis, Chlamydia trachomatis and herpes simplex virus. Skin cultures were performed on a subset of patients and controls. RESULTS: Positive prostate biopsy cultures were obtained from patients and controls. Bacteria were found in 45 of 118 pain patients (38%) and in 21 of 59 controls (36%) (p = 0.74). Older men were more likely to have positive cultures. Men with type IIIa chronic pelvic pain syndrome were more likely than those with type IIIb to have positive prostate biopsy cultures. CONCLUSIONS: Bacteria cultured from transperineal prostatic biopsies do not differ between men with and without chronic pelvic pain syndrome. Prostatic bacteria obtained by biopsy are probably not etiologically related to the symptoms in the majority of men with chronic pelvic pain syndrome.