In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women.

STUDY QUESTION: Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER: It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY: There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION: After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS: To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. WIDER IMPLICATIONS OF THE FINDINGS: This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.

PURPOSE: Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS: RNA was isolated from 5 normal human ovaries (52-79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75 years of age), and 4 PCOS ovaries (18-35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS: Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS: This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis†.

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 2, play a role in the cascade after luteinizing hormone-choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 µm), and large (>3000 µm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.