We studied the protein and mRNA expression of activin-A, follistatin and activin receptors in goat ovaries to find evidence of their possible role in ovarian activity, particularly in the various stages of follicle development. Ovaries of cyclic goats were collected and then either fixed in paraformaldehyde for immunohistochemical localisation of activin-A, follistatin, activin receptors IIA/B (ActR-IIA/B) and IA (ActR-IA) proteins or used to obtain samples to demonstrate mRNA expression of activin-A (βA subunit), follistatin, ActR-IIA, -IIB, -IA and -IB, using RT-PCR. For this latter goal, primordial, primary and secondary follicles were isolated mechanically, washed to remove the stromal cells and then used for RT-PCR. In addition, oocytes, cumulus, mural granulosa and theca cells from small (<3 mm) and large (3–6 mm) antral follicles, luteal cells and surface epithelium were collected to study mRNA expression. Activin-A and follistatin proteins were found in oocytes of all follicle classes, granulosa cells from the primary follicle stage onwards, theca cells of antral follicles, corpora lutea and ovarian surface epithelium. In antral follicles, these proteins were detected both in cumulus and mural granulosa cells. ActR-IIA/B protein was found at the same follicular sites, and also in granulosa cells of primordial follicles onward. The localisation of ActR-IA corresponded with that of ActR-IIA/B, but the former protein was absent in the theca of large antral follicles. The mRNAs for activin-A (βA subunit), follistatin, and ActR-IIA, -IIB, -IA and -IB were detected at all follicular and cellular types studied, except that ActR-IIB was not found in follicles that had not developed an antrum yet. In conclusion, in goat ovaries, transcripts of activin-A (βA subunit), its receptors and its binding protein follistatin are expressed and their proteins formed at all follicular stages and in corpora lutea. These findings indicate a role of activin-A in the local regulatory system during the entire follicular development and during luteal activity.

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

17β-Estradiol (E2) has been shown to modulate the renin–angiotensin system in hydromineral and blood pressure homeostasis mainly by attenuating angiotensin II (ANGII) actions. However, the cellular mechanisms of the interaction between E2 and angiotensin II (ANGII) and its physiological role are largely unknown. The present experiments were performed to better understand the interaction between ANGII and E2 in body fluid control in female ovariectomized (OVX) rats. The present results are the first to demonstrate that PKC/p38 MAPK signaling is involved in ANGII-induced water and sodium intake and oxytocin (OT) secretion in OVX rats. In addition, previous data from our group revealed that the ANGII-induced vasopressin (AVP) secretion requires ERK1/2 signaling. Therefore, taken together, the present observations support a novel concept that distinct intracellular ANGII signaling gives rise to distinct neurohypophyseal hormone release. Furthermore, the results show that E2 attenuates p38 MAPK phosphorylation in response to ANGII but not PKC activity in the hypothalamus and the lamina terminalis, suggesting that E2 modulates ANGII effects through the attenuation of the MAPK pathway. In conclusion, this work contributes to the further understanding of the interaction between E2 and ANGII signaling in hydromineral homeostasis, as well as it contributes to further elucidate the physiological relevance of PKC/p38 MAPK signaling on the fluid intake and neurohypophyseal release induced by ANGII.