Carbon Chain Length Determines Inhibitory Potency of Perfluoroalkyl Sulfonic Acids on Human Placental 3ß-Hydroxysteroid Dehydrogenase 1: Screening, Structure-Activity Relationship, and In Silico Analysis.

Objective: This study aimed to compare 9 perfluoroalkyl sulfonic acids (PFSA) with carbon chain lengths (C4-C12) to inhibit human placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), aromatase, and rat 3ß-HSD4 activities. Methods: Human and rat placental 3ß-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS-MS, and human aromatase activity was determined by radioimmunoassay. Results: PFSA inhibited human 3ß-HSD1 structure-dependently in the order: perfluorooctanesulfonic acid (PFOS, half-maximum inhibitory concentration, IC 50: 9.03 ± 4.83 µmol/L) > perfluorodecanesulfonic acid (PFDS, 42.52 ± 8.99 µmol/L) > perfluoroheptanesulfonic acid (PFHpS, 112.6 ± 29.39 µmol/L) > perfluorobutanesulfonic acid (PFBS) = perfluoropentanesulfonic acid (PFPS) = perfluorohexanesulfonic acid (PFHxS) = perfluorododecanesulfonic acid (PFDoS) (ineffective at 100 µmol/L). 6:2FTS (1H, 1H, 2H, 2H-perfluorooctanesulfonic acid) and 8:2FTS (1H, 1H, 2H, 2H-perfluorodecanesulfonic acid) did not inhibit human 3ß-HSD1. PFOS and PFHpS are mixed inhibitors, whereas PFDS is a competitive inhibitor. Moreover, 1-10 µmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells. Docking analysis revealed that PFSA binds to the steroid-binding site of human 3ß-HSD1 in a carbon chain length-dependent manner. All 100 µmol/L PFSA solutions did not affect rat 3ß-HSD4 and human placental aromatase activity. Conclusion: Carbon chain length determines inhibitory potency of PFSA on human placental 3ß-HSD1 in a V-shaped transition at PFOS (C8), with inhibitory potency of PFOS > PFDS > PFHpS > PFBS = PFPS = PFHxS = PFDoS = 6:2FTS = 8:2FTS.

Role of microtubule +TIPs and -TIPs in spermatogenesis - Insights from studies of toxicant models.

During spermatogenesis, preleptotene spermatocytes and haploid spermatids, lacking lamellipodia and filopodia to initiate cell movement per se, but rely on Sertoli cells for transport across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. Tracks provided by microtubules (MTs) that lay across the epithelium are essential to support germ cell and other cargo transports, but the mechanism(s) remain elusive. Studies have provided insightful information through the use of toxicant models. Herein, we summarize findings based on studies of the microtubule plus (+)-end tracking proteins (+TIPs) and the microtubule minus (-)-end targeting proteins (-TIPs), at the corresponding plus (+)-end and minus (-)-end of the polarized MTs in rat testes. We also provide a model by which + TIPs and -TIPs that work in concert with microtubule-associated proteins (MAPs; e.g., MAP-1a), MARKs (microtubule affinity-regulating kinases), and microtubule-specific motor proteins (e.g., dynein 1) to support germ cell and cargo transports. This thus provides a framework to design experiments for future studies.

Regulation of BTB Dynamics in Spermatogenesis-Insights From the Adjudin Model.

During spermatogenesis, cell organelles, and germ cells, most notably haploid spermatids, are transported across the seminiferous epithelium so that fully developed spermatids line-up at the edge of the tubule lumen to undergo spermiation at stage VIII of the cycle. Studies have suggested that the microtubule (MT)-based cytoskeleton is necessary to support these cellular events. However, the regulatory molecule(s) and underlying mechanism(s) remain poorly understood. Herein, we sought to better understand this event by using an adjudin-based animal model. Adult rats were treated with adjudin at low-dose (10 mg/kg b.w.) which by itself had no notable effects on spermatogenesis. Rats were also treated with low-dose adjudin combined with overexpression of 2 endogenously produced blood-testis barrier (BTB) modifiers, namely rpS6 (ribosomal protein S6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) and F5-peptide (a biological active peptide released from laminin-γ3 chain at the Sertoli-spermatid interface) versus the 2 BTB modifiers alone. Overexpression of these 2 BTB modifiers in the testis was shown to enhance delivery of adjudin to the testis, effectively inducing disruptive changes in MT cytoskeletons, causing truncation of MT conferred tracks that led to their collapse across the epithelium. The net result was massive germ cell exfoliation in the tubules, disrupting germ cell transport and cell adhesion across the seminiferous epithelium that led to aspermatogenesis. These changes were the result of disruptive spatial expression of several MT-based regulatory proteins. In summary, MT cytoskeleton supported by the network of MT regulatory proteins is crucial to maintain spermatogenesis.

CAMSAP2 Is a Microtubule Minus-End Targeting Protein That Regulates BTB Dynamics Through Cytoskeletal Organization.

During spermatogenesis, microtubule (MT) cytoskeleton in Sertoli cells confers blood-testis barrier (BTB) function, but the regulators and mechanisms that modulate MT dynamics remain unexplored. In this study, we examined the role of calmodulin-regulated spectrin-associated protein (CAMSAP)2 (a member of the CAMSAP/Patronin protein family), and a minus-end targeting protein (-TIP) that binds to the minus-end (i.e., slow-growing end) of polarized MTs involved in determining MT length, in Sertoli cell function. CAMSAP2 was found to localize at discrete sites across the Sertoli cell cytosol, different from end-binding protein 1 (a microtubule plus-end tracking protein that binds to the plus-end of MTs), and colocalized with MTs. CAMSAP2 displayed a stage-specific expression pattern, appearing as tracklike structures across the seminiferous epithelium in adult rat testes that lay perpendicular to the basement membrane. CAMSAP2 knockdown by RNA interference was found to promote Sertoli cell tight junction (TJ) barrier function, illustrating its role in inducing TJ remodeling under physiological conditions. To further examine the regulatory role of CAMSAP2 in BTB dynamics, we used a perfluorooctanesulfonate (PFOS)-induced Sertoli cell injury model for investigations. CAMSAP2 knockdown blocked PFOS-induced Sertoli cell injury by promoting proper distribution of BTB-associated proteins at the cell-cell interface. This effect was mediated by the ability of CAMSAP2 knockdown to block PFOS-induced disruptive organization of MTs, but also F-actin, across cell cytosol through changes in cellular distribution/localization of MT- and actin-regulatory proteins. In summary, CAMSAP2 is a regulator of MT and actin dynamics in Sertoli cells to support BTB dynamics and spermatogenesis.

Environmental toxicants and cell polarity in the testis.

During spermatogenesis, head-tail cell polarity, apico-basal cell polarity and planar cell polarity (PCP) are remarkably noted in the seminiferous epithelium in which the heads of developing haploid spermatids are pointed to the basement membrane, and with their tails to the tubule lumen. Furthermore, these polarized spermatids are laid unidirectionally across the plane of the seminiferous epithelium, mimicking PCP noted in hair cells of the inner ear. Treatment of rodents with environmental toxicants that lead to germ cell exfoliation, however, are associated with notable changes in spermatid polarity, and defects in spermatid polarity always precede spermatid loss from the epithelium. Studies have also shown that environmental toxicant-induced Sertoli cell or testis injury is mediated through changes in actin and/or microtubule (MT) cytoskeletons. Emerging evidence has illustrated that cell polarity and PCP also exert their regulatory effects through changes in cytoskeletal organization. Herein, we discuss and critically evaluate these recent findings, hoping that better efforts can be coordinated by investigators to address this rapidly developing field regarding the role of cell polarity and PCP proteins in toxicant-induced male reproductive dysfunction.