Variable occurrence of apoptosis in the testes of diploid and sterile allotetraploid Cobitis (Teleostei, Cobitidae) males during the reproductive cycle.

Cobitis species exist in both diploid and diploid-polyploid (d-p) populations, but mostly occur in the latter. They are considered an important model organism to study the biology and physiology of natural hybrid and polyploid vertebrates. Indeed, polyploidization causes a huge stress for in terms of cell physiology and alters spermatogenesis in polyploid fish. The most extensively studied mode of germ cell death during spermatogenesis in vertebrates is apoptosis. The aim of this study was to examine caspase-3 immunoexpression in the testes of Cobitis taenia from a diploid population as well as C. taenia and sterile tetraploid Cobitis from d-p populations before, during and after spawning. The obtained results suggest a different performance of apoptosis in the testes of C. taenia from the two studied populations and seems to be conditioned by their role as the only sperm donors in d-p populations. Moreover, apoptosis was an active cell death process in the testes of tetraploid Cobitis.

The Legacy of Sexual Ancestors in Phenotypic Variability, Gene Expression, and Homoeolog Regulation of Asexual Hybrids and Polyploids.

Hybridization and polyploidization are important evolutionary processes whose impacts range from the alteration of gene expression and phenotypic variation to the triggering of asexual reproduction. We investigated fishes of the Cobitis taenia-elongatoides hybrid complex, which allowed us to disentangle the direct effects of both processes, due to the co-occurrence of parental species with their diploid and triploid hybrids. Employing morphological, ecological, and RNAseq approaches, we investigated the molecular determinants of hybrid and polyploid forms. In contrast with other studies, hybridization and polyploidy induced relatively very little transgressivity. Instead, Cobitis hybrids appeared intermediate with a clear effect of genomic dosing when triploids expressed higher similarity to the parent contributing two genome sets. This dosage effect was symmetric in the germline (oocyte gene expression), interestingly though, we observed an overall bias toward C. taenia in somatic tissues and traits. At the level of individual genes, expression-level dominance vastly prevailed over additivity or transgressivity. Also, trans-regulation of gene expression was less efficient in diploid hybrids than in triploids, where the expression modulation of homoeologs derived from the "haploid" parent was stronger than those derived from the "diploid" parent. Our findings suggest that the apparent intermediacy of hybrid phenotypes results from the combination of individual genes with dominant expression rather than from simple additivity. The efficiency of cross-talk between trans-regulatory elements further appears dosage dependent. Important effects of polyploidization may thus stem from changes in relative concentrations of trans-regulatory elements and their binding sites between hybridizing genomes. Links between gene regulation and asexuality are discussed.

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.

The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERß expression and the incidence of apoptosis in granulosa cells of medium porcine follicles.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor ß (ERß) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERß and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

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.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P4) and oestradiol (E2) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P4 and stimulated E2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P4 and inhibited E2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs.

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and ß (ERα and ERß) in these cells. The influence of E2 on P4 secretion and ERα and ERß expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERß mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERß protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERß protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERß protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

The expression of the aryl hydrocarbon receptor in reproductive and neuroendocrine tissues during the estrous cycle in the pig.

The aryl hydrocarbon receptor (AhR) has been recognized as a mediator of xenobiotic-induced toxicity. In addition, it was demonstrated that the AhR is able to influence the regulation of reproductive processes in females. The aim of this study was to examine AhR mRNA (real-time PCR) and protein (Western-blot) expression in ovarian follicles and stroma, corpora lutea (CL), oviducts, endometrium, myometrium as well as in medial basal hypothalami (MBH), and anterior (AP) and posterior (PP) pituitaries harvested during the follicular (days 17-19) and luteal (days 8-10) phase of the porcine estrous cycle. The AhR transcript and protein were found in all structures collected during both phases. AhR mRNA expression tended (p=0.06) to be higher in the CL than in follicles. The AhR protein expression in ovarian stroma was higher (p≤0.01) during the follicular than in the luteal phase. Endometrial expression of AhR mRNA was higher (p≤0.01), while AhR protein was lower (p≤0.01) during the follicular phase in comparison to the luteal phase. Within neuroendocrine tissues, AhR mRNA and protein content in hypothalamus were relatively low and did not differ (p>0.05) between phases. In contrast, higher AhR mRNA expression in AP (p≤0.001) and protein expression in PP (p≤0.01) were found during the luteal phase compared to the follicular phase. Differences in AhR expression observed in reproductive and neuroendocrine tissues during the follicular and luteal phase of the estrous cycle indicate the involvement of AhR in the regulation of reproductive function in pigs.

Ciprofloxacin and furagin in acute cystitis: comparison of early immune and microbiological results.

Furagin (a nitrofurantoin analogue) has the same efficacy in treating acute cystitis as ciprofloxacin, however the duration of therapy is longer. We established a hypothesis that therapy with ciprofloxacin results in faster resolution of mucosal inflammation in comparison with furagin. Rates of urinary secretion of immunoglobulins class A, M and G and interleukin-8 (IL-8) were evaluated before and after initiation of therapy in adult women presenting with acute cystitis confirmed by urine culture. Women were randomised into two groups receiving either ciprofloxacin 250mg twice a day for 3 days (n=13) or furagin 100mg three times a day for 7 days (n=14). Median lengths of follow-up were 4 days and 5 days in the ciprofloxacin and furagin groups, respectively. Treatment with ciprofloxacin resulted in faster eradication of pathogens. No bacteria or nitrates were detected in the ciprofloxacin group, whilst leukocyte esterase was positive in only one case. In the furagin group there were four positive cultures, seven cases with positive nitrates and five cases with positive esterase. Secretion rates of all four substances dropped significantly, but the changes over time were similar in both groups.