The role of androgens in the regulation of porcine ovarian functions during neonatal and postnatal development

Androgeny regulują ekspresję genów w komórkach docelowych poprzez interakcje z receptorami androgenowymi. Dotychczasowe badania wykazały obecność receptora androgenowego oraz cytochromu P450c17 w płodowym jajniku świni, co świadczy, że jest on tkanką docelową dla androgenów lub ich antagonistów, ale także potencjalnym miejscem syntezy tych steroidów. Celem niniejszej pracy było określenie roli androgenów w regulacji funkcji jajnika świni w okresie neonatalnym i postnatalnym po zastosowaniu antyandrogenu, flutamidu, w odniesieniu do wybranych genów androgenozależnych (FSHR, Cx43, AQP5), które są istotne dla prawidłowego rozwoju i funkcjonowania jajnika. Przeprowadzone doświadczenie polegało na nastrzykiwaniu flutamidem (50 mg/kg masy ciała) ciężarnych świń w krytycznych dniach ciąży: od 20 do 28 lub od 80 do 88 dnia po zapłodnieniu, oraz prosiąt od 2 do 10 dnia po urodzeniu. Materiał do badań stanowiły jajniki pobrane od prosiąt w 2 dniu po urodzeniu oraz jajniki dojrzałych płciowo świń. Analiza morfologiczna jajników zwierząt nowonarodzonych wykazała obecność mniej zawansowanych w rozwoju struktur jajnikowych (gniazda komórek płciowych, pęcherzyki pierwotne) w stosunku do kontroli. W oparciu o analizę Real-time PCR, stwierdzono obniżoną ekspresję genów FSHR i Cx43 na poziomie mRNA, wskazując na działanie androgenów we wczesnych etapach rozwoju jajnika i folikulogenezy u świni. W pęcherzykach przedantralnych wyizolowanych z jajników dojrzałych płciowo świń stwierdzono obniżoną immunoekspresję białka Ki-67 świadczącą o ograniczeniu aktywności proliferacyjnej komórek ziarnistych. Przeprowadzona reakcja TUNEL nie potwierdziła natomiast ich apoptozy. Zmniejszona ilość transkryptu dla Cx43 oraz spadek ekspresji białka Cx43 w wyniku iniekcji flutamidu od 80 dnia ciąży i od 2 dnia po urodzeniu wskazują, że obniżony indeks proliferacji komórek ziarnistych jest ściśle związany z zaburzeniem komunikacji międzykomórkowej w pęcherzyku przedantralnym. W pęcherzykach antralnych wyizolowanych z jajników dojrzałych płciowo świń obserwowano znaczny wzrost immunoekspresji białka Ki-67, zarówno w komórkach ziarnistych, jak i komórkach osłonki wewnętrznej, we wszystkich badanych grupach. Wysoka aktywność proliferacyjna korelowała z zahamowaniem apoptozy komórek ziarnistych. Wyraźny wzrost ilości transkryptu i białka FSHR dowodzi zwiększonej stymulacji przez FSH. Wyjaśnia to podwyższoną ekspresję genu CYP19A1 oraz wzrost stężenia estradiolu w homogenatach pęcherzyków antralnych po ekspozycji na flutamid od 2 dnia po urodzeniu. Przewaga proliferacji nad apoptozą może być także wynikiem zwiększonej ekspresji mRNA i białka Cx43 po podaniu flutamidu od 80 dnia ciąży i od 2 dnia po urodzeniu. Za pomocą testu „swelling assay” stwierdzono pozytywny wpływ testosteronu na aktywność akwaporyn w komórkach ziarnistych pęcherzyków jajnikowych świni. Zaobserwowano także obniżenie ilości transkryptu i białka AQP5 w pęcherzykach antralnych po zastosowaniu flutamidu. Uzyskane wyniki dowodzą, że androgeny mogą być ważnymi regulatorami formowania antrum i płynu pęcherzykowego w pęcherzyku jajnikowym świni, poprzez wpływ na ekspresję akwaporyn. W jajniku dojrzałej płciowo świni, obok prawidłowo zbudowanych ciałek żółtych, obserwowano ciałka żółte o charakterze cyst. Neonatalne zastosowanie flutamidu spowodowało obniżenie ekspresji Cx43, 3b-HSD oraz spadek stężenia progesteronu w homogenatach ciałek żółtych. Prezentowane badania dowodzą, że komunikacja pomiędzy komórkami lutealnymi odpowiada za ich funkcje steroidogenne. Podsumowując uzyskane wyniki można stwierdzić, że zablokowanie receptora androgenowego poprzez prenatalne lub neonatalne iniekcje flutamidu wpływa na rozwój i funkcjonowanie pęcherzyków jajnikowych i ciałka żółtego u dorosłych świń.Androgens acting via androgen receptors (ARs) govern expression of various genes in target tissues. Recently, we have demonstrated the presence of ARs in the porcine fetal ovary at different days of gestation, which suggests a capacity to respond to androgens and/or antiandrogenic factors during that period. Moreover, P450c17 localized in the fetal porcine ovary indicates the potential sites of androgens synthesis. The current study was conducted to determine whether gestational or neonatal exposure to the antiandrogen flutamide influeces FSHR, Cx43 and AQP5 gene expression in the neonatal and adult porcine ovary. The androgen deficiency during the prenatal and neonatal windows may alter FSHR, Cx43 and AQP5 expression, leading to the abnormal folliculogenesis in adult pig ovaries and disturbed corpus luteum (CL) function. Flutamide (50 mg/kg body weight) was injected into pregnant gilts between days 20 and 28 or days 80 and 88 of gestation, and into female piglets between days 2 and 10 postnatally. The ovaries were collected from 2-day-old piglets or sexually mature pigs, treated and non-treated (control) ones. Following flutamide exposure during pregnancy we observed less advanced ovarian stuctures (oocyte nests, primordial follicles) in the neonatal ovaries when compared to the controls. Additionally, FSHR and Cx43 mRNA were down-regulated after flutamide exposure. These results confirm the notion that androgens play a crucial role at the early stages of porcine folliculogenesis. The diminished expression of FSHR and Cx43 in the primary follicles following late gestational flutamide treatment suggests their role in the activation of follicular development in pigs and may result in delayed folliculogenesis. In preantral follicles from adult pig ovaries, Ki-67 immunoexpression was decreased after flutamide administration. On the other hand, TUNEL reaction did not reveal any apoptotic granulosa cells. Cx43 mRNA was down-regulated and Western-blot analysis showed a decreased expression of Cx43 protein in preantral follicles. Thus, we assume a close relationship between low proliferative activity and diminished Cx43 expression. In large antral follicles from adult pig ovaries, isolated from animals following maternal and neonatal flutamide administration Ki-67 immunoexpression in granulosa and theca cells was significantly higher than in the controls. High proliferative activity correlated with a low number of apoptotic granulosa cells. Moreover, an increased mRNA and protein FSHR levels and CYP19A1 gene expression led to the elevated estradiol concentration observed following neonatal flutamide exposure. The increase in Cx43 mRNA and protein expression after late gestational and neonatal flutamide administration may also induce raised proliferative activity of granulosa cells. Furthermore, the swelling assay revealed that testosterone produced a swelling of granulosa cells in hypotonic solution, which was abolished by the anti-androgen hydroxyflutamide. In large antral follicles isolated from ovaries of animals postnatally exposed to flutamide we showed a decline of AQP5 mRNA and protein. In addition to properly formed CLs in each examined ovary, we found the cysts of corpus luteum following neonatal flutamide administration. In luteal tissues, Cx43 and 3b-HSD expression, and progesterone concentration were lower than in the controls. Overall, these results suggest substantial involvement of androgens in the regulation of pig ovarian development. This manifests by alterations of FSHR, Cx43 and AQP5 gene expression induced by administration of flutamide during particular prenatal and neonatal periods what entails disturbed porcine follicle development, as well as CL formation and function

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7) M), 2-Hf (1.7 x 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3 beta-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3 beta-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary

Prenatal exposure to antiandrogen flutamide affects androgen receptor (AR) expression in postnatal ovarian development in pig

The following study was undertaken to localize androgen receptors (ARs) in various structures of the porcine ovary after prenatal exposure to antiandrogen flutamide. In wet. treatment by antiandrogens may have adverse effects on reproductive function in immature and adult animals. Flutamide was injected into pregnant swines between days 20 and 28 (GD20) or 80 to 88 (GD80) of gestation. The ovaries were collected from treated animals and from control ones (non-treated) at two different points of development: from immature and adult pigs. Immunoexpression of AR was determined for preantral and antral follicles and for stroma cells. Immunostaining showed that AR expression in immature animals was unaffected in the primary follicles, while in the preantral and antral follicles the AR level fluctuated depending on day of treatment as well as on analyzed tissue. In adult animals, the immunoexpression of AR slightly decreased in antral follicles independently on the day of flutamide treatment. Therefore. AR expression in postnatal life may be affected by in utero exposure to antiandrogen flutamide

Effects of silver nanoparticles on proliferation and apoptosis in granulosa cells of chicken preovulatory follicles : an in vitro study

The continuous development of poultry production related to the growing demand for eggs and chicken meat makes it necessary to use modern technologies. An answer to this demand may be the use of nanotechnology in poultry farming. One of the promising nanomaterials in this field are silver nanoparticles (AgNPs), which are used as disinfectants, reducing microbial pollution and the amounts of greenhouse gases released. This study aimed to evaluate the effect of AgNPs on the proliferation and apoptosis process in the granulosa cells of chicken preovulatory follicles. The in vitro culture experiment revealed that both 13 nm and 50 nm AgNPs inhibited the proliferation of the granulosa cells. However, a faster action was observed in 50 nm AgNPs than in 13 nm ones. A size-dependent effect of AgNP was also demonstrated for the caspase-3 activity. AgNPs 13 nm in size increased the caspase-3 activity in granulosa cells, while 50 nm AgNPs did not exert an effect, which may indicate the induction of distinct cell death pathways by AgNPs. In conclusion, our study reveals that AgNPs in vitro inhibit granulosa cell proliferation and stimulate their apoptosis. These results suggest that AgNPs may disrupt the final stage of preovulatory follicle maturation and ovulation

The level of endogenous DNA damage in lymphocytes isolated from blood is associated with the fluctuation of 17\beta-estradiol concentration in the follicular phase of healthy young women

The aim of this study was to evaluate whether the differences in plasma 17β-estradiol concentration in early and late follicular phases of the menstrual cycle can affect the level of endogenous DNA damage in lymphocytes assessed by comet assay, and whether the extent of this damage in the follicular phase is associated with the genotype of catechol-O-methyltransferase (COMT). The level of DNA damage was positively correlated with 17β-estradiol concentration only in the late follicular phase. Subjects with the COMT L/L homozygous mutated variant revealed more DNA damage as compared to individuals with the COMT wild-type and heterozygous (H/L+HH) genotype