Epididymal mRNA expression profiles for the protein disulfide isomerase gene family: Modulation by development and androgens.

BACKGROUND: The endoplasmic reticulum (ER) is the central hub for protein quality control, where the protein disulfide isomerases (PDIs), encoded by at least 21 genes, play a pivotal role. These multifunctional proteins contribute to disulfide bond formation, proper folding, and protein modifications, and may act as hormone-binding proteins (e.g., steroids), influencing hormone biology. The interplay between ER proteostasis, PDIs, and epididymis-a crucial site for sperm maturation-remains largely understudied. OBJECTIVES: This study characterizes transcriptional signatures of Pdi genes in the epididymis. MATERIAL AND METHODS: Transcriptional profiles of selected Pdi genes were assessed in adult Wistar rat tissues, and epididymis under different experimental conditions (developmental stages, surgical castration, and efferent ductules ligation [EDL]). In silico bioinformatic analyses identified expression trends of this gene family in human epididymal segments. RESULTS: P4hb, Pdia3, Pdia5, Pdia6, Erp44, Erp29, and Casq1 transcripts were detected in both reproductive and non-reproductive tissues, while Casq2 exhibited higher abundance in vas deferens, prostate, and heart. Pdilt, highly expressed in testis, and Pdia2, highly expressed in heart, showed minimal mRNA levels in the epididymis. In the mesonephric duct, epididymal embryonic precursor, P4hb, Pdia3, Pdia5, Pdia6, and Erp29 mRNAs were found at gestational day (GD) 17.5. Except for Erp29, which remained stable, these Pdi transcript levels increased from GD17.5 to GD20.5, when epididymal morphogenesis occurs, and were maintained to varying degrees in the epididymis during postnatal development. Surgical castration downregulated P4hb, Pdia3, Pdia5, Pdia6, Pdilt and Erp29 transcripts, an effect reversed by testosterone replacement. Conversely, transcript levels remained unaffected by EDL, except P4hb, which was reduced in caput epididymis. All 21 PDI genes exhibited diverse transcriptional profiles across the human epididymis. DISCUSSION AND CONCLUSION: The findings lay the foundations to explore Pdi genes in epididymal biology. As a considerable proportion of male infertility cases are idiopathic, targeting hormonal regulation of protein quality control in epididymis represents a route to address male infertility and advance therapeutic interventions in this domain.

Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line.

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.

Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors.

BACKGROUND: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored. RESULTS: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells. CONCLUSIONS: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Epididymal embryonic development harbors TLR4/NFKB signaling pathway as a morphogenetic player.

The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5-20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

Novel androgen-induced activity of an antimicrobial ß-defensin: Regulation of Wolffian duct morphogenesis.

The Wolffian duct (WD) undergoes morphological changes induced by androgens to form the epididymis, which is an organ essential for sperm maturation. Androgen action in WD epithelium involves paracrine factors of mesenchymal origin that function by still poorly understood mechanisms. Here we studied the antimicrobial ß-defensin SPAG11C as a new player in duct morphogenesis, localized prenatally in the WD mesenchyme. Organotypic culture of rat WDs and tissues from Androgen Receptor (AR) knockout mice (ARKO) were used. Our results show that androgen/AR signaling differentially regulated SPAG11C expression at mRNA and protein levels in the developing WD. WDs incubated with recombinant human SPAG11C were shorter and less coiled as a result of reduced epithelial cell proliferation, but not increased apoptosis. Our results suggested ß-defensin SPAG11C as an androgen-target required for WD morphogenesis. This highlights the multifunctional repertoire of the ß-defensin protein family and their potential contribution to the in utero environment that determines male reproductive success.