A New Bioreactor to Promote Follicular Growth With or Without Activin A

Background: The aim of the present study was to evaluate the effect of Activin A on the activation of in vitro folliculogenesis of human ovarian tissues with or without our new three-dimensional structure (3D-structure). Methods: Five fresh ovarian human tissues were cultured in vitro in 4 groups with 100ng/mL Activin A or without Activine A and within or without a 3D-structure for 20 or 22 days of in vitro culture. Follicular density and quality were evaluated, and follicular diameters were measured. Estradiol secretion was quantified. Proliferation and apoptosis through immunostaining were performed.Results: The proportion of primordial follicles was significantly reduced, and the proportion of primary and secondary follicles was significantly increased in all four groups (p<0.001). Antral cavities were observed in the four culture groups. Activin A supplementation did not significantly affect the follicular density, follicular quality, follicular growth, or estradiol secretion (p>0.05). The 3D-structure increased the density of primary follicles and decreased the estradiol secretion (p<0.001). Tissular proliferation was significantly lower in the 3D-structure group compared to the non-3D-structure group (p=0.008). Regarding tissular apoptosis, it was significantly higher in the Activin group compared to the non-Activin group (0.006). Conclusion: The presence of Activin A did not seem to play a key role in in vitro folliculogenesis activation. However, the results may indicate that the 3D-structure could be more physiological and could prevent a pejorative in vitro folliculogenesis flare-up.

Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways

The purpose of this study is to evaluate the amphetamine effects on progesterone and estradiol production in rat granulosa cells and the underlying cellular regulatory mechanisms. Freshly dispersed rat granulosa cells were cultured with various test drugs in the presence of amphetamine, and the estradiol/progesterone production and the cytosolic cAMP level were measured. Additionally, the cytosolic-free Ca2+ concentrations ([Ca2+]i) were measured to examine the role of Ca2+ influx in the presence of amphetamine. Amphetamine in vitro inhibited both basal and porcine follicle-stimulating hormone-stimulated estradiol/progesterone release, and amphetamine significantly decreased steroidogenic enzyme activities. Adding 8-Bromo-cAMP did not recover the inhibitory effects of amphetamine on progesterone and estradiol release. H89 significantly decreased progesterone and estradiol basal release but failed to enhance a further amphetamine inhibitory effect. Amphetamine was capable of further suppressing the release of estradiol release under the presence of nifedipine. Pretreatment with the amphetamine for 2 h decreased the basal [Ca2+]i and prostaglandin F2α-stimulated increase of [Ca2+]i. Amphetamine inhibits progesterone and estradiol secretion in rat granulosa cells through a mechanism involving decreased PKA-downstream steroidogenic enzyme activity and L-type Ca2+ channels. Our current findings show that it is necessary to study the possibility of amphetamine perturbing reproduction in females.

Combined surgical and medical treatment in an adolescent with severe gynecomastia due to excessive estradiol secretion: a case report

Background Gynecomastia develops due to the reversed estradiol-to-Testosterone ratio in adolescence, and symptoms typically improve within 2 years. The causes vary widely, including estrogen excess and tumors, and surgical treatment is usually given in late adolescence because postoperative symptoms may recur in adolescents. This study reports a case of a pediatric patient with severe gynecomastia due to excessive estradiol secretion who showed a positive outcome after receiving surgical treatment combined with aromatase inhibitor administration. Case presentation A 9-year old boy visited to the Department of Pediatric Endocrinology for breast budding. At that time, the patient showed breasts at Tanner stage II and no abnormality on hormone tests. During a follow-up, both gynecomastia had progressed to Tanner stage III–IV at age 13. Tamoxifen 10 mg bid was administered; however, the condition rapidly progressed to Tanner stage V at 13.5 years. The evaluation of pathologic gynecomastia showed an increase of estradiol to 296 pg/mL with normal range 10 ~ 36 pg/mL and microlithiasis in both testes. As the condition worsened, total mastectomy was performed at the age of 13.5 years. Based on the assessment that elevated aromatase activity had induced breast budding, we changed the medication to anastrozole (Arimidex) 1 mg once a day, after which the estradiol level improved to 38.5 pg/mL and was maintained well in the two-year postoperative follow-up. Conclusions This case report shows a combined plastic surgery and appropriate medical management bring a positive outcome in severe gynecomastia patient.

Myostatin is expressed in bovine ovarian follicles and modulates granulosal and thecal steroidogenesis

Myostatin plays a negative role in skeletal muscle growth regulation but its potential role in the ovary has received little attention. Here, we first examined relative expression of myostatin (MSTN), myostatin receptors (ACVR1B, ACVR2B and TGFBR1) and binding protein, follistatin (FST), in granulosa (GC) and theca (TC) cells of developing bovine follicles. Secondly, using primary GC and TC cultures, we investigated whether myostatin affects steroidogenesis and cell number. Thirdly, effects of gonadotropins and other factors on MSTN expression in GC and TC were examined. MSTN, ACVR1B, TGFBR1, ACVR2B and FST mRNA was detected in both GC and TC at all follicle stages. Immunohistochemistry confirmed follicular expression of myostatin protein. Interestingly, MSTN mRNA expression was lowest in GC of large estrogen-active follicles while GC FST expression was maximal at this stage. In GC, myostatin increased basal CYP19A1 expression and estradiol secretion whilst decreasing basal and FSH-induced HSD3B1 expression and progesterone secretion and increasing cell number. Myostatin also reduced IGF-induced progesterone secretion. FSH and dihydrotestosterone had no effect on granulosal MSTN expression whilst insulin-like growth factor and tumour necrosis factor-alpha suppressed MSTN level. In TC, myostatin suppressed basal and LH-stimulated androgen secretion in a follistatin-reversible manner and increased cell number, without affecting progesterone secretion. LH reduced thecal MSTN expression whilst BMP6 had no effect. Collectively, results indicate that, in addition to being potentially responsive to muscle-derived myostatin from the circulation, myostatin may have an intra-ovarian autocrine/paracrine role to modulate thecal and granulosal steroidogenesis and cell proliferation/survival.