Małgorzata Kotula-Balak

Expression of connexin 43 protein in testes, epididymides and prostates of stallions

REASONS FOR PERFORMING STUDY Connexin 43 (Cx43) is a ubiquitously distributed gap junction protein in testes and other reproductive tissues. Adjacent cells share ions and small metabolites through intercellular channels, which are present in gap junctions. Previously, Cx43 has not been reported in testes, epididymides and prostates either in healthy stallions or cryptorchid horses. OBJECTIVES To demonstrate the expression pattern of Cx43 in the reproductive tissues of stallions and examine whether naturally occurring bilateral cryptorchidism has any influence on distribution and expression of Cx43. METHODS The expression and the presence of Cx43 protein were detected by means of immunohistochemistry and Western blot analysis using a polyclonal rabbit anti-Cx43 antibody. RESULTS In stallions, gap junctions appeared as structures localised to cell-cell contacts between adjacent cells. In testes, Cx43 expression was detected in the interstitial tissue and seminiferous tubules, between Leydig and Sertoli, as well as Sertoli and germ cells. In epididymides, Cx43 was localised between epithelial cells, whereas in prostates, between secretory cells of the glandular epithelium. In the cryptorchid, a clear reduction of Cx43 signal was observed in all reproductive tissues. CONCLUSIONS Coupling of Leydig cells via gap junctions may suggest that steroidogenic function of the testis is under the influence of these intercellular channels. Within seminiferous tubules, the expression was found to be stage-specific, pointing to its role in coordinating spermatogenesis. Differential distribution of Cx43 protein in the reproductive tract of normal and cryptorchid stallions indicates that expression is clearly dependent on the physiological status of the horse. POTENTIAL RELEVANCE Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.

Relaxin family peptides in the male reproductive system—a critical appraisal

The human genome project has identified, besides ovarian relaxin (RLN), six other relaxin-like molecules (RLN3, H1-RLN, INSL3-6), most of which appear to be expressed in the testis and/or male reproductive system, together with four different G-protein-coupled receptors responsive to one or other of these peptides. Earlier work on relaxin in the male assumed the simplistic hypothesis of only a single relaxin-like entity. This review systematically examines the expression and physiology of relaxin-like molecules in the male reproductive system in order to reappraise the importance of this hormone system for male reproductive function. Although there are important species differences, only INSL3 and INSL6 appear to be generally expressed at a moderately high level within the testis, whereas ovarian RLN is consistently a major secretory product of the prostate epithelium. However, all members of this relaxin-like family appear to be expressed also at a low level in different organs of the male reproductive system, suggesting possible autocrine/paracrine effects. The four receptors (RXFP1-4) for these peptides are also expressed to differing levels in both somatic and seminiferous compartments of the testis and in the prostate, supporting relevant functions for most members of this interesting peptide family. Recent studies of relaxin family peptides in prostate pathology highlight their functional importance in the clinical context as potential causative, diagnostic and therapeutic agents and warrant more specific and detailed studies of their roles also in regard to male fertility and other aspects of male reproductive function.

Androgen aromatization in cryptorchid mouse testis

Estrogens play an important role in germ cell development. Therefore, we have studied expression patterns of aromatase that converts testosterone into estrogens in 2 recombinant inbred mouse strains that differ in efficiency of spermatogenesis. In order to show whether germ cells are a target for estrogens, estrogen receptors (ER)alpha and beta were localized as well. Adult male CBA and KE mice were made unilaterally cryptorchid to determine alterations in testicular steroidogenesis and spermatogenesis. Differences between control and cryptorchid testes have been studied with respect to (1) cellular sites of aromatase, the enzyme responsible for estrogen formation, (2) the presence of ERalpha and ERbeta in various types of testicular cells, and (3) steroidogenic activity in the testes. Additionally, unilaterally control testes of cryptorchid mice were compared with bilaterally descended testes. Histological or hormonal differences were not found between control testes of cryptorchid and untreated mice. In cryptorchid testes from both strains, degeneration of germ cells was observed as well as a decrease in size of the seminiferous tubules, whereas the amount of interstitial tissue increased, especially in testes of CBA mice. Using immunohistochemistry, aromatase was localized in Leydig cells and germ cells in both control and cryptorchid testes. Sertoli cells were immunopositive in control testes only. In cryptorchid testes of KE mice, aromatase was strongly expressed in spermatids, that were still present in a few tubules. Other cell types in tubules were negative for aromatase. In both control and cryptorchid testes of both mouse strains, ERalpha were present in Leydig cells only, whereas ERbeta were found in Leydig cells and in germ cells in early stages of maturation. In homogenates of testes of CBA control mice, testosterone levels were 3-fold higher than in those of control KE mice, whereas the difference in estradiol levels between both strains was small. Cryptorchidism resulted in decreased testosterone levels and increased estradiol levels. The results of the present study show functional alterations due to cryptorchidism in both mouse strains. Strong aromatase expression in germ cells in control and cryptorchid testes indicates an additional source of estrogens in the testis besides the interstitial tissue and the relevance of estrogen in spermatogenesis.

Effects of 4-tert-octylphenol on the testes and seminal vesicles in adult male bank voles

The present study was designed to evaluate the effects of 4-tert-octylphenol (OP) on male testes and seminal vesicles of bank vole. Adult males kept under long or short photoperiod were orally administered OP (200mg/kg bw) for 30 or 60 days. Treatment for 30 days had no discernible effect on the parameters examined. Treatment for 60 days adversely influenced weights and histological structure of the testes and seminal vesicles. In these tissues, expression of 3β-hydroxysteroid dehydrogenase and androgen receptor and testosterone levels were reduced, whereas expression of aromatase and estrogen receptor α and estradiol levels were increased. The alterations were more evident in voles kept in long photoperiod. Taken together, it is suggested that adverse changes in bank vole reproductive tissues induced by long-term OP-exposure result from disturbed androgen and estrogen synthesis and action. Moreover, there might be a subtle difference in the sensitivity to OP between voles kept in different light conditions.

Connexin 43 expression in human and mouse testes with impaired spermatogenesis

Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelters syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelters patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelters patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar

In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg(-1) d(-1) was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen-estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.

Morphological and functional alterations in adult boar epididymis: Effects of prenatal and postnatal administration of flutamide

BackgroundThe dynamic cross-talk between epididymal cells is hormonally regulated and, in part, through direct cell-to-cell interactions. To date, no information is available regarding possible impact of anti-androgens on the proteins involved in the gap junctional communication within the boar epididymis. Thus, a question arised whether prenatal or postnatal exposure to an anti-androgen flutamide alters the expression of gap junction protein - connexin43 (Cx43) and androgen receptor (AR) expression in the caput, corpus and cauda epididymis and leads to delayed effects on morphology and function of adult pig epididymis.MethodsFirst two experimental groups received flutamide prenatally on gestational days 20-28 and 80-88 (GD20 and GD80) and further two groups were exposed to flutamide postanatally on days 2-10 and 90-98 after birth (PD2 and PD90). Epididymides were collected from adult boars. Routine histology was performed using hematoxylin-eosin staining. The expression of Cx43 and AR were analyzed using immunohistochemistry and Western blotting. Both analyses were supported by quantitative approaches to demonstrate the variations of the expression levels following the treatment. Apoptotic cells were identified using TUNEL assay.ResultsHistological examination revealed differences in epididymal morphology of flutamide-exposed boars when compared to controls. Scarce spermatic content were seen within the corpus and cauda lumina of GD20, PD2 and PD90 groups. Concomitantly, frequency of epididymal cell apoptosis was significantly higher (p < 0.05) after exposure to flutamide at GD20. Moreover, in GD20, PD2, and PD90 groups, significantly lower AR expression (p < 0.05) was found in the principal and basal cells of the corpus and cauda regions, while in the stromal cells AR expression was significantly reduced (p < 0.05) along the epididymal duct. Concomitantly, a decrease in Cx43 expression (p < 0.05) was noticed in the stromal cells of the cauda region of GD20 and PD2 groups. This indicates high sensitivity of the stromal cells to androgen withdrawal.ConclusionsThe region-specific alterations in the epididymis morphology and scarce spermatic content within the lumina of the corpus and cauda indicate that flutamide can induce delayed effects on the epididymal function of the adult boar by decrease in AR protein levels that results in altered androgen signaling. This may cause disturbances in androgen-dependent processes including Cx43 (de)regulation, however, we can not exclude the possibility that in response to flutamide decreased Cx43 expression may represent one mechanism responsible for functional disturbance of the boar epididymis.

Does 4-tert-octylphenol affect estrogen signaling pathways in bank vole Leydig cells and tumor mouse Leydig cells in vitro?

Primary Leydig cells obtained from bank vole testes and the established tumor Leydig cell line (MA-10) have been used to explore the effects of 4-tert-octylphenol (OP). Leydig cells were treated with two concentrations of OP (10(-4) M, 10(-8) M) alone or concomitantly with anti-estrogen ICI 182,780 (1 μM). In OP-treated bank vole Leydig cells, inhomogeneous staining of estrogen receptor α (ERα) within cell nuclei was found, whereas it was of various intensity among MA-10 Leydig cells. The expression of ERα mRNA and protein decreased in both primary and immortalized Leydig cells independently of OP dose. ICI partially reversed these effects at mRNA level while at protein level abrogation was found only in vole cells. Dissimilar action of OP on cAMP and androgen production was also observed. This study provides further evidence that OP shows estrogenic properties acting on Leydig cells. However, its effect is diverse depending on the cellular origin.

Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system

Seminal plasma of rainbow trout (Oncorhynchus mykiss, Salmonidae) contains an inhibitory system consisting of three fractions (I-III) characterized by different electrophoretic migration rates. Using a two-step isolation procedure we purified (20- and 43-fold to homogeneity) and characterized the two subforms of inhibitor I (Ia and Ib). On the basis of the homology alignment of the amino acid sequences, inhibitor I was classified to the family of cysteine proteinase inhibitors - fetuins. The molecular masses were determined to be 61,146.5Da and 63,096.0Da, and the isoelectric points were estimated to be 6.04 and 6.22 for inhibitor Ia and Ib. Both inhibitors were glycoproteins with a carbohydrate content about 13% for inhibitor Ia and 19% for inhibitor Ib. The equilibrium association constant of inhibitor Ib with cod trypsin was determined to be 7.1×10(8)M(-1). Except for the cod trypsin inhibition, the inhibitor Ib effectively inhibited papain belonging to the cysteine proteainases. Comparative studies of the distribution of inhibitor I and the previously described inhibitor II were performed. The presence of inhibitor I in the seminal plasma was a common feature of several Salmoniformes, which was contrary to inhibitor II detected in seminal plasma of other fish families. Inhibitors I and II showed different expression patterns in the testes and spermatic duct of the rainbow trout.

Identification of parvalbumin-like protein as a major protein of common carp (Cyprinus carpio L) spermatozoa which appears during final stage of spermatogenesis

Parvalbumin is well known as the major fish allergen that is typically present in high amounts in muscles, where it functions in calcium buffering and is involved in the relaxation process in fast-twitch muscles. We show in our current study that parvalbumin-like protein is present in high amounts in carp spermatozoa. It is the first report to demonstrate the presence of parvalbumin-like protein in fish spermatozoa. Using antibodies produced against purified carp parvalbumin-like protein, we localized parvalbumin-like protein to spermatids and spermatozoa. Our results indicate that parvalbumin-like protein appeared during the final stage of spermatogenesis. We also detected high amounts of parvalbumin-like protein in carp seminal plasma but not in blood plasma which suggests that its function may be specific for the male reproductive tract. The activation mechanism of carp sperm movement is not fully understood, but in carp, Ca2+ influx is the prerequisite for the initiation of sperm motility. The appearance of parvalbumin-like protein in high amounts in mature spermatozoa coincides with their acquiring the ability to move. The presence of parvalbumin-like protein in spermatozoa and seminal plasma strongly suggests that parvalbumin-like protein is an important part of the Ca2+-mediated mechanism of sperm activation in carp.