Anna Leska

Seasonal changes in the expression of the androgen receptor in the testes of the domestic goose (Anser anser f. domestica)

It is generally acknowledged that seasonal fluctuations in the morphology and function of bird testes are primarily regulated by seasonal changes in circulating concentrations of testosterone (T) which mediates its action via the androgen receptor (AR). However, it has not yet been elucidated whether gonadal sensitivity to androgens also varies across the bird reproductive cycle. In order to answer the above question, this study makes the first ever attempt to account for the gonadal expression of the AR gene and protein in relation to circulating and testicular T concentrations in the gonads of male birds during the reproductive cycle. The experimental model used in this study was the domestic goose, Anser anser f. domestica, a species with three distinct phases of the annual reproductive cycle: the breeding season in March, the non-breeding season in July and the sexual reactivation phase in November. The plasma and testicular T concentrations were highest in the breeding season, followed by a dramatic decline in the non-breeding season with a successive rise in the sexual reactivation phase. Interestingly, we observed the divergent effect of season on AR mRNA and protein expression. Whereas the AR gene expression showed a nearly inverse relationship with T levels, the seasonal variations in AR protein levels primarily reflected the differences in T concentrations. The results of our study also indicated that regardless of the examined phase of the season, an abundance of AR protein was found only in the nuclei of Leydig and Sertoli cells and myoid cells. The above supports the observation that somatic cells are the targets for androgen action in bird testes. Summarizing, this study revealed that seasonal variations in sensitivity to androgens in the gonads of male birds are reflected in variations in the availability of their cognate receptors. Furthermore, a different pattern of seasonal expression of the AR gene and protein suggests that the AR system is subject to complex regulation that includes both steroid-dependent and steroid-independent factors.

Estradiol concentration and the expression of estrogen receptors in the testes of the domestic goose (Anser anser f. domestica) during the annual reproductive cycle.

Seasonal fluctuations in the activity of bird testes are regulated by a complex mechanism where androgens play a key role. Until recently, the role played by estrogens in males has been significantly underestimated. However, there is growing evidence that the proper functioning of the testes is associated with optimal estradiol (E2) concentration in both the plasma and testes of many mammalian species. Estrogens are gradually emerging as very important players in hormonal regulation of reproductive processes in male mammals. Despite the previously mentioned, it should be noted that estrogenic action is limited by the availability of specific receptors--estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Interestingly, there is a general scarcity of information concerning the estrogen responsive system in the testes of male birds, which is of particular interest in exploring the phenomenon of seasonality of reproduction. To address this question, we have investigated for the first time the simultaneous expression of testicular ERα and ERβ genes and proteins with the accompanying plasma and testicular E2 concentrations during the annual reproductive cycle of male bird. The research model was the domestic goose (Anser anser f. domestica), a species whose annual reproductive cycle can be divided into 3 distinct phases characterized by changes in testicular activity. It has been revealed that the stable plasma E2 profile did not correspond to changing intratesticular E2 profile throughout the experiment. The expression of ERα and ERβ genes and proteins was detected in gander testes and it fluctuated on a seasonal basis with lower level in breeding and sexual reactivation stages and higher level during the nonbreeding stage. Our results demonstrated changes in testicular sensitivity to estrogens in male domestic goose during the annual reproductive cycle. The seasonal pattern of estrogen receptors (ERs) expression was analyzed against the hormonal background and a potential mechanism of ERs regulation in bird testes was proposed. The present study revealed seasonal variations in the estrogen responsive system, but further research is needed to fully explore the role of estrogens in the reproductive tract of male birds.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae)

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2–5 and 10–15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Expression of the androgen receptor in the testes and the concentrations of gonadotropins and sex steroid hormones in male turkeys (Meleagris gallopavo) during growth and development.

Androgens, including testosterone (T) and androstenedione (A4), are essential for puberty, fertility and sexual functions. The biological activity of those hormones is mediated via the androgen receptor (AR). The regulation of androgen action in birds is poorly understood. Therefore, the present study analysed mRNA and protein expression of AR in the testes, plasma concentrations of the luteinizing hormone (LH), follicle-stimulating hormone (FSH), T, A4 and oestradiol (E2), as well as the levels of T, A4 and E2 in testicular homogenates of male turkeys (Meleagris gallopavo) at the age of 4, 8, 12, 16, 20, 24 and 28weeks. Plasma concentrations of LH and FSH, as well as plasma and testicular levels of T and A4 began to increase at 20weeks of age. The lowest plasma levels of E2 were noted at 20weeks relative to other growth stages. The 20th week of life seems to be the key phase in the development of the reproductive system of turkeys. The AR protein was found in the nuclei of testicular cells in all examined growth stages. Higher expression of AR protein in the testes beginning at 20weeks of age was accompanied by high plasma concentrations of LH and high plasma and testicular levels of androgens. This relationship seems to be necessary to regulate male sexual function.

Mechanism of phytoestrogen action in Leydig cells of ganders (Anser anser domesticus): Interaction with estrogen receptors and steroidogenic enzymes

Phytoestrogens (PE) are plant-derived compounds that have an estrogen-like activity and they can influence male and female reproduction. The possible mechanisms of PE action may be including: the binding to estrogen receptors (ER) and the interaction with the key steroidogenic enzymes. The aim of this study was to investigate if PE has effect on steroidogenesis of gander testicular cells by above-described pathways. The Leydig cells were isolated from testes of White Kołuda® ganders at the peak of their reproductive activity (March). These Leydig cells (1×105per mL) were pre-incubated with the ER inhibitor – ICI 182, 780 (100 nM) for 3 h and then these cells were incubated with PE (5 and 50 μM): genistein, daidzein, equol and coumestrol during next 20 h or untreated control and the Leydig cells that were previously treated (20 h) with genistein (5 and 50 μM) were incubated for next 6 h with steroid intermediates (20 μM) as testosterone (T) precursors: hydroxycholesterol, pregnenolone, progesterone and androstenedione. Concentrations of T in the samples of incubation medium were measured using radioimmunoassay. Genistein, daidzein, and equol (5 and 50 μM) decreased (P < 0.05) T secretion by incubated gander Leydig cells and ICI 182, 780 did not eliminate the inhibitory effect of these PE. After genistein (50 μM) treatment, basal and stimulated with 22R-hydroxycholesterol, pregnenolone, progesterone and androstenedione, T production by testicular cells was decreased (P < 0.05). In contrast, genistein at lower dose (5 μM) did not affect the stimulatory effects of testosterone precursors. In conclusion, the inhibition of testosterone secretion by the phytoestrogens in gander Leydig cells did not depend on estrogen receptors. The suppression of steroidogenesis in these cells may be in part conducted by interaction of phytoestrogens with key steroidogenic enzymes. However, further studies are required to elucidate the phytoestrogen mechanism of action in gander testicular cells.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.