Perfluorooctanesulfonate (PFOS) Perturbs Male Rat Sertoli Cell Blood-Testis Barrier Function by Affecting F-Actin Organization via p-FAK-Tyr407: An in Vitro Study

Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10–20 μM (∼5–10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr407 was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr407 was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr407 and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB.

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Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Reproduction ◽

10.1530/rep-06-0385 ◽

2007 ◽

Vol 133 (6) ◽

pp. 1169-1179 ◽

Cited By ~ 97

Author(s):

Tu’uhevaha J Kaitu’u-Lino ◽

Pavel Sluka ◽

Caroline F H Foo ◽

Peter G Stanton

Keyword(s):

Mrna Expression ◽

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Cell Contacts ◽

Barrier Formation ◽

Protein Components ◽

Blood Testis Barrier

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJsin vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P< 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesisin vivois partly via their effects on TJ proteins and regulation of the blood–testis barrier.

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116. Contribution of claudin-11 to the inter-Sertoli cell tight junction, in vitro

Reproduction Fertility and Development ◽

10.1071/srb05abs116 ◽

2005 ◽

Vol 17 (9) ◽

pp. 72

Author(s):

M. J. McCabe ◽

P. G. Stanton

Keyword(s):

Tight Junction ◽

Mrna Expression ◽

Sertoli Cell ◽

Adhesion Molecule ◽

Sertoli Cells ◽

Adherens Junction ◽

Adherens Junction Protein ◽

Junction Protein

The inter-Sertoli cell tight junction (TJ) forms the blood testis barrier (BTB) between Sertoli cells and is composed of three major transmembrane proteins: claudin-11, occludin and junctional adhesion molecule. Formation of the BTB occurs during puberty associating with an increase in circulating gonadotrophins. Claudin-11 and occludin are hormonally regulated in vitro although their importance to the function of the TJ is unknown. The aim of this study was to investigate the contribution of claudin-11 to the inter-Sertoli cell TJ in vitro by blocking gene expression using RNA interference. Two claudin-11-specific siRNA fragments were designed for this purpose. Sertoli cells in primary culture formed stable TJs within 5 days as measured by transepithelial electrical resistance (TER). The addition of siRNA for 2 days resulted in a significant (P < 0.01) 55% (mean, SD, n = 4 cultures) decrease in TER along with a major reduction in claudin-11 localisation to the TJ as assessed by immunocytochemistry. The specificity of the siRNA was shown by the presence of extensive immunostaining of occludin and of the adherens junction protein β-catenin in the same treatments. Similarly, claudin-11 mRNA expression significantly (P < 0.01) decreased by 71% (mean, SD, n = 3 cultures) in response to both claudin-11 siRNA fragments. Occludin mRNA expression was not affected. It is concluded that claudin-11 contributes at least 55% to the function of the rat Sertoli cell TJ in vitro. It is hypothesised that the remaining 45% of TJ function can be attributed to other integral proteins, such as occludin and junctional adhesion molecule. It is expected that claudin-11 and other TJ proteins play a pivotal role in the function of the BTB in vivo with potential implications in fertility and contraception.

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rpS6 Regulates Blood-Testis Barrier Dynamics By Affecting F-Actin Organization and Protein Recruitment

Endocrinology ◽

10.1210/en.2012-1665 ◽

2012 ◽

Vol 153 (10) ◽

pp. 5036-5048 ◽

Cited By ~ 45

Author(s):

Ka-Wai Mok ◽

Dolores D. Mruk ◽

Bruno Silvestrini ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Actin Filament ◽

Sertoli Cells ◽

Plasma Membranes ◽

Permeability Barrier ◽

Actin Organization ◽

Filament Bundles ◽

Mtorc1 Pathway ◽

Protein Recruitment ◽

Blood Testis Barrier

Abstract During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII–IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB.

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Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00176.2014 ◽

2014 ◽

Vol 307 (7) ◽

pp. E553-E562 ◽

Cited By ~ 19

Author(s):

Xiang Xiao ◽

Dolores D. Mruk ◽

Elissa W. P. Wong ◽

Will M. Lee ◽

Daishu Han ◽

...

Keyword(s):

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Protein Trafficking ◽

In Vitro Study ◽

Underlying Mechanism ◽

Endocytic Vesicle ◽

Immunological Barrier ◽

Blood Testis Barrier

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the “old” BTB located above the preleptotene spermatocytes being transported across the immunological barrier is “disassembled,” whereas the “new” BTB found behind these germ cells is rapidly “reassembled,” i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed.

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311. Adult Sertoli cells proliferate in response to exogenous follicle stimulating hormone in the adult photo-inhibited Djungarian hamster

Reproduction Fertility and Development ◽

10.1071/srb05abs311 ◽

2005 ◽

Vol 17 (9) ◽

pp. 133

Author(s):

G. A. Tarulli ◽

P. G. Stanton ◽

S. J. Meachem

Keyword(s):

Cell Proliferation ◽

Tight Junction ◽

Sertoli Cell ◽

Cell Population ◽

Sertoli Cells ◽

Follicle Stimulating Hormone ◽

Tight Junction Proteins ◽

Djungarian Hamster ◽

Junction Proteins ◽

Blood Testis Barrier

Sperm production relies on nutritional and structural support from Sertoli cells. Sertoli cells undergo maturational changes (e.g. cessation of proliferation and formation of the blood–testis barrier) around the onset of puberty in higher mammals1 and maturational failure has been associated with some infertility syndromes and testicular malignancies2. The Sertoli cell population is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the adult Djungarian hamster showed that Sertoli cell numbers decreased by 35% in the absence of serum gonadotrophins, and returned to control levels by short-term replacement of follicle stimulating hormone (FSH)3. Therefore, the aims of this study were to (i) quantify the proliferative activity of Sertoli cells in the hormonally manipulated Djungarian hamster, and (ii) examine the localisation of several tight junction proteins as markers of the blood–testis barrier. Long day (LD) photoperiod (16L : 8D) adult hamsters were exposed to short day (SD) photoperiod (8L : 16D) for 11 weeks to suppress gonadotrophins and then received FSH for up to 10 days. Sertoli cell proliferation was assessed immunohistochemically by the colocalisation of GATA-4 and PCNA, and quantified by stereology. Tight junction proteins (occludin and ZO-1) were colocalised using confocal microscopy. Sertoli cell proliferation in both the LD and SD controls was minimal; however, in response to FSH treatment proliferation was upregulated within 4 days compared with SD controls (98% v. 2%, P < 0.001, respectively). Tight junction proteins colocalised at the blood–testis barrier in LD hamsters, but were disorganised within the Sertoli cell cytoplasm in SD animals. FSH treatment restores colocalisation in a time-dependent manner. It is concluded that FSH contributes to the regulation of Sertoli cell proliferation and tight junction formation in the adult Djungarian hamster. This data provides definitive evidence that the adult Sertoli cell population in this model is modifiable by hormones. (1)Meachem et al. (2005). Biol Reprod 72, 1187.(2)Allan et al. (2004). Endocrinol 145, 1587.(3)Russell and Peterson (1985). Int Rev Cytol 94, 177.

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002.Sertoli cell terminal differentiation: doing a 'U' turn on a one way street

Reproduction Fertility and Development ◽

10.1071/srb05abs002 ◽

2005 ◽

Vol 17 (9) ◽

pp. 62

Author(s):

S. J. Meachem

Keyword(s):

Cell Proliferation ◽

Tight Junction ◽

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Proliferative Activity ◽

Terminal Differentiation ◽

Tight Junction Proteins ◽

Junction Proteins ◽

Blood Testis Barrier

The concept of terminal differentiation of Sertoli cells has been challenged and this new information has important implications for male fertility. The mammalian Sertoli cell has two distinct functions: (i) formation of the seminiferous cords and (ii) provision of nutritional and structural support to the developing germ cells. For these to occur successfully, Sertoli cells must undergo numerous maturational changes between foetal and adult life, the main switches occur around the onset of puberty, coincident with the rise in serum gonadotrophins. These switches include the loss of proliferative activity and the formation of the blood testis barrier. Follicle stimulating hormone (FSH) plays a key role in supporting Sertoli cell proliferation in early postnatal life and thus is critical in establishing sperm output in adulthood. After puberty, the size of the Sertoli cell population is considered to be stable and unmodifiable by hormones. This accepted view has been contested as data shows that the size of the adult Sertoli cell population is modifiable by hormone suppression, and that Sertoli cells can regain proliferative activity when stimulated by FSH in the Djungarian hamster1. The molecular mechanism(s) by which Sertoli cells re-enter proliferation are not known in this model however a study demonstrated that helix-loop-inhibitor of differentiation proteins can induce terminally differentiated Sertoli cells to re-enter the cell cycle and proliferate2. Thyroid hormone and testosterone may be involved in the cessation of Sertoli cell proliferation. Gonadotrophin suppression in the adult Djungarian hamster also results in the disruption of the blood testis barrier and spatial organisation of the inter Sertoli cell tight junction proteins and as a consequence the loss of all germ cells that reside inside the blood testis barrier. FSH restores the organisation of these tight junction proteins, which is associated with the appearance of more mature germ cells. It is expected that the integrity of the blood testis barrier is also re-established. It is suggested that this demonstrated plasticity of the adult Sertoli cell may be relevant in clinical settings, particularly to some types of infertility and testicular malignancies where Sertoli cells have failed to undergo these important maturational switches. (1)Chaudhary et al. (2005) Biol. Reprod. 72, 1205. (2)Meachem et al. (2005) Biol. Reprod. 72, 1187.

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Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽

10.1210/en.2014-1163 ◽

2014 ◽

Vol 155 (10) ◽

pp. 3981-3995 ◽

Cited By ~ 19

Author(s):

N. Ece Gungor-Ordueri ◽

Elizabeth I. Tang ◽

Ciler Celik-Ozenci ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Adherens Junction ◽

Actin Binding ◽

Permeability Barrier ◽

Tight Junction Permeability ◽

Residual Bodies ◽

Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

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CX43 expression, phosphorylation, and distribution in the normal and autoimmune orchitic testis with a look at gap junctions joining germ cell to germ cell

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00500.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. R121-R139 ◽

Cited By ~ 13

Author(s):

R.-Marc Pelletier ◽

Casimir D. Akpovi ◽

Li Chen ◽

Robert Day ◽

María L. Vitale

Keyword(s):

Cell Differentiation ◽

Gap Junctions ◽

Germ Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Seminiferous Tubule ◽

Connexin 43 ◽

Cx43 Expression ◽

Cx43 Protein ◽

Cx43 Mrna

Spermatogenesis requires connexin 43 (Cx43).This study examines normal gene transcription, translation, and phosphorylation of Cx43 to define its role on germ cell growth and Sertoli cell's differentiation, and identifies abnormalities arising from spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and a natural model for autoimmunity. Northern blot analysis detected 2.8- and a 3.7-kb Cx43 mRNA bands in seminiferous tubule-enriched fractions. Cx43 mRNA increased in seminiferous tubule-enriched fractions throughout development and then seasonally with the completion of spermatogenesis. Cx43 protein levels increased transiently during the colonization of the tubules by the early-stage spermatocytes. Cx43 phosphorylated (PCx43) and nonphosphorylated (NPCx43) in Ser368 decreased during the periods of completion of meiosis and Sertoli cell differentiation, while Cx43 mRNA remained elevated throughout. PCx43 labeled chiefly the plasma membrane except by stage VII when vesicles were also labeled in Sertoli cells. Vesicles and lysosomes in Sertoli cells and the Golgi apparatus in the round spermatids were NPCx43 positive. A decrease in Cx43 gene expression was matched by a Cx43 protein increase in the early, not the late, phase of AIO. Total Cx43 and PCx43 decreased with the advance of orchitis. The study makes a novel finding of gap junctions connecting germ cells. The data indicate that Cx43 protein expression and phosphorylation in Ser368 are stage-specific events that may locally influence the acquisition of meiotic competence and the Sertoli cell differentiation in normal testis. AIO modifies Cx43 levels, suggesting changes in Cx43-mediated intercommunication and spermatogenic activity in response to cytokines imbalances in Sertoli cells.

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Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

Biochemical Journal ◽

10.1042/bj3400309 ◽

1999 ◽

Vol 340 (1) ◽

pp. 309-320 ◽

Cited By ~ 11

Author(s):

Sikha Bettina MUKHERJEE ◽

S. ARAVINDA ◽

B. GOPALAKRISHNAN ◽

Sushma NAGPAL ◽

Dinakar M. SALUNKE ◽

...

Keyword(s):

Cell Culture ◽

Germ Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Culture Media ◽

Glutathione S Transferase ◽

Steroid Binding ◽

Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

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148. HORMONALLY REGULATED miRNAS TARGET THE TUBULOBULBAR COMPLEX IN THE TESTIS

Reproduction Fertility and Development ◽

10.1071/srb10abs148 ◽

2010 ◽

Vol 22 (9) ◽

pp. 66

Author(s):

P. K. Nicholls ◽

P. G. Stanton ◽

K. L. Walton ◽

R. I. McLachlan ◽

L. O'Donnell ◽

...

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Focal Adhesions ◽

Intercellular Junctions ◽

Protein Translation ◽

Seminiferous Epithelium ◽

Seminiferous Tubules ◽

Micro Rnas

Spermatogenesis is absolutely dependent on follicle stimulating hormone (FSH) and androgens; acute suppression of these hormones inhibits germ cell development and thus sperm production. The removal of intercellular junctions and release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to acute hormone suppression(1). To define the molecular mechanisms that mediate FSH and androgen effects in the testis, we investigated the expression and hormonal regulation of micro-RNAs (miRNA), small non-coding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs that were upregulated >2-fold in stage VIII seminiferous tubules following hormone suppression, and in vitro in primary Sertoli cells. We subsequently validated the expression and hormonal regulation of several miRNAs, including miR-23b, -30d and -690 by quantitative PCR in primary Sertoli cells. Bioinformatic analysis of potential targets of hormonally-suppressed miRNAs identified genes associated with Focal adhesions (54 genes, P = –ln(17.97)) and the Regulation of the actin cytoskeleton (52 genes, P = –ln(10.16)), processes known to be intimately associated with adhesion of spermatids to Sertoli cells(2, 3). Furthermore, this analysis identified numerous components of the testicular tubulobulbar complex (TBC) as being targets of hormonally sensitive miRNAs. The TBC is a podosome-like structure between Sertoli and adjacent spermatids in the testis, which internalises intact inter-cellular junctions by endocytotic mechanisms prior to spermiation(4). We then demonstrate the hormonal regulation of predicted miRNA target proteins, and validate novel inhibitory miRNA interactions with Pten, nWASP, Eps15 and Picalm by luciferase knockdown in vitro. We hypothesise that hormonally suppressed miRNAs inhibit TBC function, and subsequently, endocytosis of intercellular junctions. In conclusion, we have demonstrated that hormonal suppression in the testis stimulates the expression of a subset of Sertoli cell miRNAs that are likely regulators of cell adhesion protein networks involved in spermiation. (1) Saito K, O’Donnell L, McLachlan RI, Robertson DM 2000 Spermiation failure is a major contributor to early spermatogenic suppression caused by hormone withdrawal in adult rats. Endocrinology 141: 2779–2.(2) O’Donnell L, Stanton PG, Bartles JR, Robertson DM 2000 Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat. Biol Reprod 63: 99–108.(3) Beardsley A, Robertson DM, O’Donnell L 2006 A complex containing alpha6beta1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium. J Endocrinol 190(3): 759–70.(4) Young JS, Guttman JA, Vaid KS, Vogl AW 2009 Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis. Biol Reprod 80: 162–74.

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