Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health

Adjudin-mediated junction restructuring in the seminiferous epithelium leads to displacement of soluble guanylate cyclase from adherens junctions

A plethora of evidence supports the role of cyclic nucleotides in junction restructuring. For instance, studies have shown cGMP to be a key regulator of junction assembly and disassembly in different in vitro and in vivo systems. In this study, we examine the role of soluble guanylate cyclase (sGC) in junction restructuring in the seminiferous epithelium of the rat testis. First, the interaction of soluble guanylate cyclase β1 (sGCβ1; sGC is a heterodimer comprised of an α and a β subunit) with proteins that constitute adherens and tight junctions in the testis was demonstrated. By immunoprecipitation, sGCβ1 was found to associate with occludin, JAM-A, and ZO-1, as well as with cadherin, catenin, nectin, afadin, ponsin, and espin, suggestive of its role in cell junction dynamics. These results were corroborated in part by immunohistochemistry experiments, which revealed that the localization of sGCβ1 was largely restricted to the site of the apical and basal ectoplasmic specialization. Next, the role of sGC in junction dynamics was addressed by using an in vivo model of junction restructuring. Administration of Adjudin-a chemical entity known to specifically perturb adhesion between Sertoli and germ cells (i.e., round and elongate(ing) spermatids and most spermatocytes)-resulted in a ≃1.5-fold increase in sGCβ1, coinciding with the loss of germ cells from the epithelium. More importantly, the ability of sGCβ1 to associate with cadherin increased approximately three-fold during Adjudin-mediated restructuring of Sertoli-germ cell junctions, whereas its interaction with tight junction proteins (i.e., occludin and ZO-1) decreased. Taken collectively, these results suggest that sGC participates in the remodeling of cell junctions during spermatogenesis

Nitric oxide-cGMP signaling: Its role in cell junction dynamics during spermatogenesis

During spermatogenesis, development of spermatogonia into elongated spermatids takes place in the seminiferous epithelium of the adult mammalian testis. Specifically, post-meiotic germ cell maturation occurs in a unique microenvironment sequestered from the systemic circulation by the blood-testis barrier (BTB), which is formed by adjacent Sertoli cells. Therefore, an intact BTB, as ell as stable Sertoli-germ cell adhesion, are important criteria for successful spermatogenesis. To date, numerous factors have been shown to influence spermatogenesis, and among them is the well-studied nitric oxide (.NO)/guanosine 3′,5′-cyclic monophosphate (cGMP) signaling cascade. The enzymes of this pathway, namely nitric oxide synthase, soluble guanylate cyclase and cGMP-dependent protein kinase, have all been shown to regulate cell junctions in the testis. Likewise, recent findings have shown that this signaling cascade also plays a critical role in the regulation of Sertoli-germ cell adhesion. In this mini-review, we briefly discuss the regulatory role of each protein component of the NO/ cGMP pathway in the context of testicular junction dynamics, as well as their importance in fertility and male contraception

Rab4A GTPase-catenin interactions are involved in cell junction dynamics in the testis

A plethora of evidence has recently accumulated to suggest that Rab guanosine triphosphates (GTPases) may have functions other than those originally proposed in vesicle formation, movement, docking, and fusion. Studies have shown, for example, that Rab proteins interact with actin filaments and microtubules, illustrating cross-talk between intracellular transport and cytoskeletal dynamics. In this report, we show that Rab4A associates with adherens junction signaling proteins in the testis. By immunoprecipitation, Rab4A was found to interact with α- and β-catenin as well as with actin, vimentin, α- and β-tubulin, and protein kinase C (PKC)-α and -ε. Additionally, administration of Adjudin to adult rats upregulated the Rab4A level, which coincided with the loss of spermatocytes, round and elongating/elongated spermatids from the seminiferous epithelium. More importantly, the ability of Rab4A to associate with α- and β-catenin increased during Adjudin-induced junction restructuring in the testis, illustrating that Rab4A-catenin interactions are likely to be involved in the disassembly of Sertoli-germ cell contacts. Taken collectively, these results suggest that Rab4A participates in adherens junction dynamics

Interleukin 1 alpha (IL1A) is a novel regulator of the blood-testis barrier in the rat

Throughout spermatogenesis, leptotene spermatocytes must traverse the blood-testis barrier (BTB) at stages VIII-XI to gain entry into the adluminal compartment for continued development. However, the mechanism underlying BTB restructuring remains somewhat elusive. In this study, interleukin 1 alpha (IL1A) was administered intratesticularly to adult rats in order to assess its effects on spermatogenesis. IL1A was shown to perturb Sertoli-germ cell adhesion, resulting in germ cell loss from ∼50% of seminiferous tubules by 15 days posttreatment. Equally important, the functional integrity of the BTB was compromised when inulin-fluorescein isothiocyanate was detected in the adluminal compartment of the seminiferous epithelium following its administration via the jugular vein. Interestingly, IL1A did not affect the steady-state levels of proteins that confer BTB function, namely OCLN, CLDN1, F11R, TJP1, and CDH2. Instead, the localizations of OCLN, F11R, and TJP1 in the seminiferous epithelium were altered; these proteins appeared to move away from sites of cell-cell contact. Moreover, IL1A was shown to perturb the orderly arrangement of filamentous actin at the BTB and apical ectoplasmic specialization with distinct areas illustrating loss of actin filaments. Taken collectively, these results suggest that IL1A-induced BTB disruption is not mediated via the reduction of target protein levels. Instead, IL1A\u27s primary cellular target appears to be the Sertoli cell actin cytoskeleton. It is possible that localized production of IL1A by Sertoli and/or germ cells in vivo results in BTB restructuring, and this may facilitate the movement of leptotene spermatocytes across the BTB

Effect of SNAP on superoxide anion (O2⁻) production and NADPH oxidase activity in VSMC from 12-week old SHR and age-matched WKY rats.

<p>Confluent VSMC from 12-week-old SHR and age-matched WKY rats were incubated in the absence (control) and presence of SNAP (100 μM) for 24 h and O₂⁻ production (A) and NADPH oxidase activity (B) were determined as described in Materials and methods. Values are mean ± SD of 5 separate experiments using different cell populations from different animals. *P<0.05, ***P<0.001 vs WKY CTL group; ^#P<0.05 vs SHR CTL group.</p

Schematic diagram summarizing the possible mechanisms by which SNAP attenuates the enhanced expression of Giα proteins and hyperproliferation of VSMC.

<p>Schematic diagram summarizing the possible mechanisms by which SNAP attenuates the enhanced expression of Giα proteins and hyperproliferation of VSMC.</p

Effect of SNAP on expression c-Src phosphorylation in VSMC from 12-week old SHR and age-matched WKY rats.

<p>Confluent VSMC were incubated in the absence (control) or presence of SNAP (100 μM) for 24 h. The cell lysates were prepared and subjected to Western blot analysis using specific antibodies against phosphorylated c-Src (upper panels) as described in the Materials and methods. Quantification of protein bands was done by densitometric scanning (lower panels). The results are expressed as a percentage of the WKY control (CTL), taken as 100%. Values are the mean ± SD of 4 separate experiments using different cell populations from different animals. *P<0.05, ** P<0.01 vs WKY CTL group, ^## P<0.001 vs SHR CTL group.</p

Effect of SNAP on the AT1 receptor expression in the aortic vascular smooth muscle cells (VSMC) from 12-week old spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats.

<p>Confluent VSMCs were starved 24 h and incubated in the absence (control) or presence of SNAP (100 μM) for 24 h. The cell lysates were prepared and subjected to Western blot analysis using specific antibodies against AT1 (N-10) or dynein as loading control, (upper panel) as described in the Materials and methods. Quantification of protein bands was done by densitometric scanning (lower panel). The results are expressed as a percentage of the WKY control (CTL), taken as 100%. Values are the mean ± SD of 4 separate experiments using different cell populations from different animals.* P<0.05, *** P<0.001 vs WKY CTL group, ^### P<0.001 vs SHR CTL group.</p