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STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors’ ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors’ functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The “heteromer hypothesis” explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

Background: Consumption of nuts particularly walnuts has been widely recommended for lowering the risk of coronary heart disease; however, very few studies have evaluated the potential unfavourable effects that Walnut seeds might have in vivo. Objective: To investigate the long-term effects of Juglans regia seed supplementation in specially formulated feed when fed to developed mice model. Method: C57BL/6J male mice were divided into four groups, labelled, C (control), T (High Fat Simple Carbohydrate- HFSC), Cw (control + walnut) and Tw (HFSC+ walnut) based on diet. Four mice from each group were sacrificed at the end of the first and fifth month respectively. Blood samples were collected every month. Bleeding time, blood platelet number and morphology were studied. Histopathological analysis of heart and aorta were also performed. Results: Groups Cw and Tw showed a significant increase in platelet count; however, platelet activation was not detected. There was a trend to an increase in bleeding time in group Cw. Expanded lumen diameter of the aorta was observed in Cw and Tw mice initially, however, it was found to constrict by the end of the study. A large amount of peripheral fat deposition in the aortas of Cw and Tw was observed, which increased through the course of the experiment. Conclusion: Kashmiri walnut seed supplementation was found to reduce the signs in shape change of platelets as well as increase platelet number with a trend to an increased bleeding time. This observation could be of medical interest. Histopathological analysis of heart and aorta showed lipid sequestration which merits further investigations.

Given the association between subclinical hypothyroidism and metabolic syndrome, we wanted to explore if high-fat, simple-carbohydrate (HFSC) diet affects hypothalamus–pituitary–thyroid axis. One-month-old male C57BL/6J mice were fed with control (C) and HFSC (T) feed (n = 18 each), respectively, for 5 months. There was a significant increase in triiodothyronine in the T group (13.5%) compared with the age-matched C group by the fifth month. Thyroid-stimulating hormone was significantly higher (1 month: 1.9-fold; 3 months: 2.66-fold; 5 months: 3.5-fold) from the first to fifth months in the T group compared with age-matched C group. Thyrotropin-releasing hormone (TRH) gene expression showed significant decrease (1 month: 83.2%; 5 months: 40.7%) in the T group compared with the age-matched C group. TRHR1 showed significant decrease in the T group compared with the age-matched C group throughout the study (1 month: 82.8%; 3 months: 45.7%; 5 months: 75.2%). However, TRHR2 showed dynamic change during the study. Initially there was significant (1 month: 0.104-fold) downregulation, followed by significant upregulation (3 months: 3.6-fold) and downregulation (0.73-fold) by the fifth month in the T group compared with the age-matched C group. There was marked depletion of functional follicular cells and colloid substance in the thyroid glands of the T group by the fifth month compared with the C group. Leptin receptors ObRa (1 month: 48.25%; 5 months: 88%) and ObRb (1 month: 46.9%; 5 months: 63.3%) were significantly downregulated in the T group compared with the age-matched C group in the first and fifth months of feeding the respective diets. The expression of p-STAT3, a transcription factor known to have a role in energy balance, intermediate metabolism, and leptin signalling was seen to decrease significantly (6.25-fold) in the hypothalamus of the T group compared with the age-matched C group. In conclusion, HFSC feed disrupts the hypothalamus–pituitary–thyroid axis in male C57BL/6J mice.