Milena Doroszko

Functional Expression of FSH Receptor in Endometriotic Lesions.

CONTEXT FSH receptor (FSHR), besides being expressed in gonads, is also expressed in some extragonadal tissues at low levels. OBJECTIVE We examined the functional expression of FSHR in different types of endometriotic lesions. DESIGN Extensive studies were carried out to detect functional FSHR expression and FSH-stimulated estrogen production in ovarian endometriomas and recto-vaginal endometriotic nodules (RVEN). Normal endometrium, ovary, and myometrium tissues from nonpregnant cycling women served as controls. SETTINGS This laboratory-based study was carried out on tissue specimens from patients with endometriosis and healthy donors. RESULTS Endometriotic lesions and normal secretory-phase endometrium showed FSHR expression at both mRNA and protein level. RVEN and ovarian endometrioma demonstrated up-regulated CYP19A1, dependent on the activation of CYP19A1 proximal promoter II. Estrogen receptor-β (ESR2) expression was significantly increased in RVEN vs normal endometrium. Recombinant human FSH stimulation of RVEN explants significantly increased estradiol production and CYP19A1 and ESR2 expression. FSHR was up-regulated in recombinant human FSH-stimulated endometrial and decidualized stromal cells with increased CYP19A1 expression. CONCLUSIONS We described a novel functional FSHR expression, where FSH-stimulated CYP19A1 expression and estrogen production in RVEN are demonstrated. This locally FSH-induced estrogen production may contribute to the pathology, development, progression, and severity of RVEN.

Revisiting the expression and function of follicle-stimulation hormone receptor in human umbilical vein endothelial cells

Expression of follicle-stimulation hormone receptor (FSHR) is confined to gonads and at low levels to some extragonadal tissues like human umbilical vein endothelial cells (HUVEC). FSH-FSHR signaling was shown to promote HUVEC angiogenesis and thereafter suggested to have an influential role in pregnancy. We revisited hereby the expression and functionality of FSHR in HUVECs angiogenesis, and were unable to reproduce the FSHR expression in human umbilical cord, HUVECs or immortalized HUVECs (HUV-ST). Positive controls as granulosa cells and HEK293 cells stably transfected with human FSHR cDNA expressed FSHR signal. In contrast to positive control VEGF, FSH treatment showed no effects on tube formation, nitric oxide production, wound healing or cell proliferation in HUVEC/HUV-ST. Thus, it remains open whether the FSH-FSHR activation has a direct regulatory role in the angiogenesis of HUVECs.

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor–null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr–/– mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution.

Functional Implications of LH/hCG Receptors in Pregnancy-Induced Cushing Syndrome

Context: Elevated human choriogonadotropin (hCG) may stimulate aberrantly expressed luteinizing hormone (LH)/hCG receptor (LHCGR) in adrenal glands, resulting in pregnancy-induced bilateral macronodular adrenal hyperplasia and transient Cushing syndrome (CS). Objective: To determine the role of LHCGR in transient, pregnancy-induced CS. Design, Setting, Patient, and Intervention: We investigated the functional implications of LHCGRs in a patient presenting, at a tertiary referral center, with repeated pregnancy-induced CS with bilateral adrenal hyperplasia, resolving after parturition. Main Outcome Measures and Results: Acute testing for aberrant hormone receptors was negative except for arginine vasopressin (AVP)–increased cortisol secretion. Long-term hCG stimulation induced hypercortisolism, which was unsuppressed by dexamethasone. Postadrenalectomy histopathology demonstrated steroidogenically active adrenocortical hyperplasia and ectopic cortical cell clusters in the medulla. Quantitative polymerase chain reaction showed upregulated expression of LHCGR, transcription factors GATA4, ZFPM2, and proopiomelanocortin (POMC), AVP receptors (AVPRs) AVPR1A and AVPR2, and downregulated melanocortin 2 receptor (MC2R) vs control adrenals. LHCGR was localized in subcapsular, zona glomerulosa, and hyperplastic cells. Single adrenocorticotropic hormone–positive medullary cells were demonstrated in the zona reticularis. The role of adrenal adrenocorticotropic hormone was considered negligible due to downregulated MC2R. Coexpression of CYP11B1/CYP11B2 and AVPR1A/AVPR2 was observed in ectopic cortical cells in the medulla. hCG stimulation of the patient’s adrenal cell cultures significantly increased cyclic adenosine monophosphate, corticosterone, 11-deoxycortisol, cortisol, and androstenedione production. CTNNB1, PRKAR1A, ARMC5, and PRKACA gene mutational analyses were negative. Conclusion: Nongenetic, transient, somatic mutation-independent, pregnancy-induced CS was due to hCG-stimulated transformation of LHCGR-positive undifferentiated subcapsular cells (presumably adrenocortical progenitors) into LHCGR-positive hyperplastic cortical cells. These cells respond to hCG stimulation with cortisol secretion. Without the ligand, they persist with aberrant LHCGR expression and the ability to respond to the same stimulus.

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice

Background/Aims: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. Methods: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. Results: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. Conclusion: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.

Distinct testicular steroidogenic response mechanisms between neonatal and adult heat-acclimated male rats.

Background: In comparison to short-term gonad heat exposure, little is known about the molecular mechanisms that regulate testicular steroidogenesis during long-term whole body heat acclimation. Material and Methods: Testicular slices from neonatal (NHA) and adult (AHA) heat-acclimated Wistar rats were analysed in vitro to assess the mRNA expression and enzymatic activity of steroidogenic enzymes under basal and luteinising hormone (LH) or prolactin (PRL) stimulated conditions compared with control rats (CR). Furthermore, a de-acclimated group (DA) was created by transferring adult NHA rats to control conditions. Results: Heat acclimation significantly increased plasma LH levels in the AHA group and LH and PRL in the NHA group compared with the CR group; however, after heat acclimation, the T and E2 levels did not differ from the control levels. All heat-acclimated groups showed high basal intra-testicular steroid production in vitro. Moreover, basal Cyp11a1 and Hsd3b1 levels were upregulated in vitro in the NHA and DA groups versus the CR group. LH in vitro stimulation upregulated Cyp11a1 expression in the NHA and AHA groups and PRL stimulation upregulated Cyp17a1 levels in the NHA and DA groups compared with the basal expression levels. In the AHA group, decreased basal Star and CYP11A activities but increased HSD3B1 and CYP17A1 activities were found. Conclusion: Our data revealed that despite the similar steroid levels in plasma and secreted in vitro by neonatal and adult heat-acclimated rat testicular slices, the molecular mechanisms underlying the steroidogenic response to heat acclimation during these different developmental stages were distinct.

Transgenic mice expressing inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen (Tag) transgene as a model for the therapy of granulosa cell-derived ovarian cancer

Granulosa cell tumors are rare, 3-7.6% of primary ovarian tumors, although with poor prognosis as the tumor-related mortality rate is 37.3%, with 80% of deaths occurring on recurrence. We have created a transgenic (TG) murine model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian Virus 40 T-antigen (Tag), under the regulation of murine inhibin α-subunit 6 kb promoter (inhα/Tag). Gonadotropin dependent ovarian granulosa cell tumors were formed in females by the age of 5-6 months, with a 100% penetrance. We have successfully used the inhα/Tag model to test different treatment strategies for ovarian tumors. With a gene therapy trial in inhα/Tag mice crossbred with inhα/HSV-TK (herpes simplex virus thymidine kinase) mice (double TG), we proved the principle that targeted expression of HSV-TK gene in gonadal somatic cell tumors enabled tumor ablation by anti-herpes treatment. When we aimed at targeted destruction of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) expressing inhα/Tag tumor cells in vivo by a lytic peptide Hecate-CGβ conjugate, we could successfully kill the tumor cells, sparing the normal cells. We recently found high zona pellucida glycoprotein 3 (ZP3) expression in inhα/Tag granulosa cell tumors, as well as in human granulosa cell tumors. We tested the concept of treating the ovarian tumors of inhα/Tag mice by vaccination against the ectopically expressed ZP3. Immunotherapy with recombinant human (rh) ZP3 was highly successful with no objective side effects in inhα/Tag females, suggesting rhZP3 immunization as a novel strategy for the immunotherapy of ovarian granulosa cell tumors.