Evidence that activin A directly modulates early human male germline differentiation status

Abstract

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFβ superfamily ligands; this study tests the hypothesis that, activin A, a TGFβ superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25–25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFβ1, and TGFβ2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

The Impact of Activin A on Fetal Gonocytes: Chronic Versus Acute Exposure Outcomes

“…Kit was significantly lower following activin A exposure (0.85-fold), and significantly higher following SB431542 (1.75-fold), and Tdgf1, encoding the Nodal co-receptor, was significantly reduced by activin A (Figure 3A). The mRNA encoding the Nodal inhibitor Lefty2 is a known activin A-responsive gene, demonstrated in mouse embryonic stem cells and P19 embryonic carcinoma cells (52,53), and in human TCam-2 cells (30). This responsiveness was also demonstrated here in isolated gonocytes, with a 1.61-fold increase in Lefty2 following activin A exposure (Figure 3A).…”

“…Previous cell culture experiments with mouse postnatal day 6 and 15 Sertoli cells revealed SMADs localise to the nucleus following exposure to 5 ng/mL activin A (29). Further, the human germ cell-like line, TCam-2, is responsive to activin A at 5 ng/mL (30). However, the local concentration of activin A in the fetal testis is unknown, therefore we determined the optimum concentration of activin A by culturing whole testes with 0 (vehicle control; 4 µM HCl), 5, 25, 50 or 100 ng/mL human recombinant activin A (R&D Systems, Minneapolis, MN, USA) and activin-responsive genes measured.…”

“…In each well (48-well plate), 20,000 germ cells were added in 500 µL media (MEM-a, 10% FBS, 1% penicillinstreptomycin) containing 10 µM SB431542, 5 ng/mL activin A, or relevant vehicle control. A lower concentration of activin A was used compared with the whole testis cultures, as cells grown as a monolayer have been demonstrated to be robustly responsive to 5 ng/mL activin A (29,30). Cells were cultured for 24 hrs in 5% CO 2 /air, after which the cells were collected for transcript analysis.…”

“…A recent study identified that activin A promotes a less differentiated transcript profile in the human germ cell-like cell line, TCam-2 (30). To determine if germline differentiation was similarly altered in Inhba KO mice, we examined early and differentiation-associated germ cell transcripts in the Inhba WT and KO RNA-Seq dataset.…”

Epigenetics, ovarian cell plasticity, and platelet-rich plasma: Mechanistic theories

“…In adult human ovarian tissue, characterization of Piwi‐piRNA is preliminary, although basic aspects have already been discovered in testes ( Moody et al 2020 ). Present in hematopoietic stem cells and certain malignancies, Piwi-piRNA engages to produce essentially unbounded proliferation and renewal.…”