The “Male Pill”: The Learning Curve from Basic Science to the Drug Development Pipeline and the Strength of Interdisciplinary Collaboration

Scientifically Led National Enterprises PanelNearly five decades have elapsed since the development of the female contraceptive hormone pill. During this time numerous alternative female contraceptive methods have also gained acceptance, however “the pill” and other hormone-based female contraceptive methods remain the most widely used. There are many couples where the female cannot use existing methods. For the male, effective contraceptive methods are limited to condoms and vasectomy. NIH, WHO, and the National Academy of Science Institute of Medicine have all stressed the need to develop new non-hormonal contraceptive methods. In the US, 30% of men use these male methods, so there is already a significant acceptance of male alternatives for actively participating in family planning. My basic research in male reproductive biology has always focused on identifying regulators of sperm or testis function that could be employed to develop a male contraceptive

A Method for Preparation, Storage and Activation of Large Populations of Immotile Sea Urchin Sperm

Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32 P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCI, allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCI concentration (10 mM)

Development of a Novel Space Flight Plan to Monitor Female Mice Fertility Using Reduced Crew Time

Ovarian estrogen impacts the normal homeostatic and metabolic processes of all tissues and organ systems within the body: particularly, but not limited to canonical space-flight impacted systems: bone, muscle, immune, wound repair, and cardiovascular. Effects of space flight on the ovarian estrogen production are therefore critical to our understanding of all space flight experiments using female mice, the current paradigm being used on the International Space Station (ISS). Recently, we demonstrated that vaginal wall histology could be used to determine the stage of the estrous cycle in female mice at the time of sacrifice in space. Moreover, this robust technique was completed following two post-flight freezethaw procedures of the carcasses (RR1 experiment). Thus, this technique represents a viable mechanism to determine the estrous cycle status of the female at the time of sacrifice and can be completed in a manner that does not impact primary experimental objectives. We propose that vaginal wall histology become a standard procedure completed on all mice sacrificed in space and that the individual estrous status of each animal be shared with all investigators. While evidence of estrous cyclicity was present in long-term (33 day) RR1 mice, fertility of female mice exposed to weightlessness remains unknown. In preparation for an upcoming funded NASA flight investigating the effects of long duration spaceflight on female fertility, we have refined our experimental design to minimize crew flight time and to accommodate the duration of Dragon capsule berth. These refinements maintain all our proposed primary and secondary experimental objectives. Briefly, in order to evaluate fertility, we will super ovulate mice using standard procedures (PMSG hCG), followed by collection of reproductive tract after follicular stimulation alone (PMSG) or following ovulation (hCG). Ovarian folliculogenesis and ovulation rate will be determined in fixed tissues following return in order to determine fertility. Ovarian and uterine tissues will also be evaluated by hormonal and gene expression profiling using quantitative approaches (radioimmunoassays, western blots, digital droplet PCR). Comparisons will be made to contemporary vivarium and Rodent Research Hardware Transporter and Habitat housed animals maintained on earth. Supported by NNX15AB48G to JST

Synthesis and Evaluation of Eight- and Four-membered Iminosugar Analogues as Inhibitors of Testicular Ceramide-specific Glucosyltransferase, Testicular β-Glucosidase 2, and other Glycosidases

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve: ringclosing metathesis and Sharpless asymmetric dihydroxylation, and for the four-membered analogues: Sharpless epoxidation, epoxide ring opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6), was moderately active against rat-derived ceramide-specific glucosyltransferase and four of the other eight-membered analogues were weakly active against rat-derived β-glucosidase 2. Among the four-membered analogues, ((2R,3s,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25), displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonyl-azetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of β-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived β-glucosidase and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC50 of 600 nM and β-glucosidase (almond) with an IC50 of 20 µM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied

A Tissue Retrieval and Postharvest Processing Regimen for Rodent Reproductive Tissues Compatible with Long-Term Storage on the International Space Station and Postflight Biospecimen Sharing Program

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at −80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA’s Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities

Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands