F5-Peptide and mTORC1/rpS6 Effectively Enhance BTB Transport Function in the Testis—Lesson From the Adjudin Model

During spermatogenesis, the blood–testis barrier (BTB) undergoes cyclic remodeling that is crucial to support the transport of preleptotene spermatocytes across the immunological barrier at stage VIII to IX of the epithelial cycle. Studies have shown that this timely remodeling of the BTB is supported by several endogenously produced barrier modifiers across the seminiferous epithelium, which include the F5-peptide and the ribosomal protein S6 [rpS6; a downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1)] signaling protein. Herein, F5-peptide and a quadruple phosphomimetic (and constitutively active) mutant of rpS6 [i.e., phosphorylated (p-)rpS6-MT] that are capable of inducing reversible immunological barrier remodeling, by making the barrier “leaky” transiently, were used for their overexpression in the testis to induce BTB opening. We sought to examine whether this facilitated the crossing of the nonhormonal male contraceptive adjudin at the BTB when administered by oral gavage, thereby effectively improving its BTB transport to induce germ cell adhesion and aspermatogenesis. Indeed, it was shown that combined overexpression of F5-peptide and p-rpS6-MT and a low dose of adjudin, which by itself had no noticeable effects on spermatogenesis, was capable of perturbing the organization of actin- and microtubule (MT)-based cytoskeletons through changes in the spatial expression of actin- and MT-binding/regulatory proteins to the corresponding cytoskeleton. These findings thus illustrate the possibility of delivering drugs to any target organ behind a blood–tissue barrier by modifying the tight junction permeability barrier using endogenously produced barrier modifiers based on findings from this adjudin animal model.

Cryoprotectant agents and cooling effect on embryos of Macrobrachium amazonicum

SummaryThere are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.

Condition of larvae of western rock lobster (Panulirus cygnus) in cyclonic and anticyclonic eddies of the Leeuwin Current off Western Australia

Changes in the offshore oceanographic processes are suspected to be the cause of a recent dramatic decline in the settlement of post-larvae of the Western Australian spiny lobster (Panulirus cygnus), which has greatly reduced the productivity from the world’s second-largest spiny lobster fishery. The present study assessed whether there are differences in the nutritional condition of the larvae of P. cygnus sampled from two pairs of cyclonic eddies (CEs) and anticyclonic eddies (AEs). Morphometric and biochemical analyses were undertaken on the mid–late-stage larvae (VI, VII, VIII) sampled offshore from two pairs of adjacent counter-rotating mesoscale eddies in the Leeuwin Current off Western Australia. The results showed that larvae captured from CEs had greater average total dry mass (P<0.001) than those from AEs. Stage VIII larvae from CEs contained more protein (P<0.008) (38.5% ±5.4s.e.) and lipid (P<0.005) (67.2% ±12.1) than did those from AEs. The possible causes for these differences are uncertain but may be related to differences in water temperatures in CEs v. AEs influencing the ability of phyllosomas to accumulate nutritional reserves.

Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation.

p-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr397, a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII–X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr397 that regulates spermatid adhesion at the apical ES in vivo.

MAP/microtubule affinity-regulating kinases, microtubule dynamics, and spermatogenesis

During spermatogenesis, spermatids derived from meiosis simultaneously undergo extensive morphological transformation, to become highly specialized and metabolically quiescent cells, and transport across the seminiferous epithelium. Spermatids are also transported back-and-forth across the seminiferous epithelium during the epithelial cycle until they line up at the luminal edge of the tubule to prepare for spermiation at stage VIII of the cycle. Spermatid transport thus requires the intricate coordination of the cytoskeletons in Sertoli cells (SCs) as spermatids are nonmotile cells lacking the ultrastructures of lamellipodia and filopodia, as well as the organized components of the cytoskeletons. In the course of preparing this brief review, we were surprised to see that, except for some earlier eminent morphological studies, little is known about the regulation of the microtubule (MT) cytoskeleton and the coordination of MT with the actin-based cytoskeleton to regulate spermatid transport during the epithelia cycle, illustrating that this is a largely neglected area of research in the field. Herein, we summarize recent findings in the field regarding the significance of actin- and tubulin-based cytoskeletons in SCs that support spermatid transport; we also highlight specific areas of research that deserve attention in future studies.

Sertoli–germ cell junctions in the testis: a review of recent data

Spermatogenesis is a process that involves an array of cellular and biochemical events, collectively culminating in the formation of haploid spermatids from diploid precursor cells known as spermatogonia. As germ cells differentiate from spermatogonia into elongated spermatids, they also progressively migrate across the entire length of the seminiferous epithelium until they reach the luminal edge in anticipation of spermiation at late stage VIII of spermatogenesis. At the same time, these germ cells must maintain stable attachment with Sertoli cells via testis-unique intermediate filament- (i.e. desmosome-like junctions) and actin- (i.e. ectoplasmic specializations, ESs) based cell junctions to prevent sloughing of immature germ cells from the seminiferous epithelium, which may result in infertility. In essence, both desmosome-like junctions and basal ESs are known to coexist between Sertoli cells at the level of the blood–testis barrier where they cofunction with the well-studied tight junction in maintaining the immunological barrier. However, the type of anchoring device that is present between Sertoli and germ cells depends on the developmental stage of the germ cell, i.e. desmosome-like junctions are present between Sertoli and germ cells up to, but not including, step 8 spermatids after which this junction type is replaced by the apical ES. While little is known about the biology of the desmosome-like junction in the testis, we have a relatively good understanding of the molecular architecture and the regulation of the ES. Here, we discuss recent findings relating to these two junction types in the testis, highlighting prospective areas that should be investigated in future studies.

Tight junctions in the testis: new perspectives

In the testis, tight junctions (TJs) are found between adjacent Sertoli cells at the level of the blood–testis barrier (BTB) where they coexist with basal ectoplasmic specializations and desmosome-gap junctions. The BTB physically divides the seminiferous epithelium into two distinct compartments: a basal compartment where spermatogonia and early spermatocytes are found, and an adluminal compartment where more developed germ cells are sequestered from the systemic circulation. In order for germ cells (i.e. preleptotene spermatocytes) to enter the adluminal compartment, they must cross the BTB, a cellular event requiring the participation of several molecules and signalling pathways. Still, it is not completely understood how preleptotene spermatocytes traverse the BTB at stage VIII of the seminiferous epithelial cycle. In this review, we discuss largely how TJ proteins are exploited by viruses and cancer cells to cross endothelial and epithelial cells. We also discuss how this information may apply to future studies investigating the movement of preleptotene spermatocytes across the BTB.

Claudin and occludin expression and function in the seminiferous epithelium

Integral membrane proteins that contribute to function of the blood–testes barrier (BTB) in mice include claudins 3, 5 and 11 and occludin. Although claudin 11 is expressed throughout all stages of the seminiferous epithelial cycle, claudins 3 and 5 have specific expression at stage VIII. These differences in protein expression suggest that the interactions among, and functions of, these integral membrane proteins may shift over the course of the seminiferous epithelial cycle. Also, differences in expression among rodent species and men may make interpretation of studies across species challenging. This review will discuss the characteristics of claudins and occludin; the expression, regulation and function of these integral membrane proteins in the seminiferous epithelium; and how these properties relate to the unique features of BTB.

Dynamin II interacts with the cadherin- and occludin-based protein complexes at the blood–testis barrier in adult rat testes

In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.