Gene expression profiles of neonatal porcine Sertoli cells at baseline and after incubation in normal human serum as determined by RNA sequencing

ABSTRACT

Sertoli cells are unique cells that contribute to the formation of the blood-testis barrier, which is important in sustaining the environment to promote spermatogenesis and to protect immunogenic germ cells from autoimmune destruction. This is achieved through tight junctions and production of regulatory immune factors. These Sertoli cell attributes make them a relevant model for various studies involving male reproduction, autoimmune protection, and even transplantation. RNA sequencing analyses were performed on baseline neonatal porcine Sertoli cells (NPSC) and NPSC after incubation in normal human serum for 90 minutes. We previously analyzed this data for immune-related factors, such as complement components, and for differentially expressed genes related to immune function. Still, these data sets provide insight into understanding how Sertoli cells create an immunoregulatory environment, which has applications in reproduction, transplantation, and autoimmunity.

KEYWORDS:

• blood-testis barrier
• complement
• Sertoli cells

Introduction

Sertoli cells are located within the seminiferous tubules of the testis where they form the blood-testis-barrier (BTB). This tissue barrier creates a unique environment within the seminiferous tubules conducive to spermatogenesis. It acts to protect the maturing germ cells from autoimmune responses. Sertoli cells contribute to the function of the BTB by creating a physical barrier made of junctional complexes, controlling the transport of molecules necessary for spermatogenesis, and producing immune regulatory factors. This latter attribute has led to the usage of Sertoli cells as a model of immune protection in transplantation^1,2.

Our lab has conducted xenotransplantation studies utilizing neonatal porcine Sertoli cells (NPSC)^3–5, as transgenic pigs are now a clinically relevant source of xenotransplantable tissue as demonstrated with a patient receiving a pig heart in 2022⁶. We performed RNA sequencing on NPSC at baseline and NPSC after 1.5 h incubation in normal human serum (NHS) to identify complement inhibitory factors expressed by NPSC and to investigate changes in gene expression that may be affected by exposure to xenogeneic antibodies and complement^7–9, as humoral antibody-mediated activation of complement is a key mechanism of xenograft rejection¹⁰. Likewise, this data set is relevant in the study of immune protection and regulation.

Methods

Testes collection

A detailed overview of the methods used in this data note is contained in our previously published works by Washburn et. al., 2023^8,9. Briefly, testes (n = 24) were collected from three-to-five-day old Duroc-Landrace pigs (n = 12) by the Texas Tech University New Deal Swine Unit, which maintained all animals in adherence to the approved Institute for Laboratory Animal Research Care (IACUC), Use of Laboratory Animals, Texas Tech University Health Sciences Center’s guidelines and protocols of the National Institutes of Health (protocol number 05019).

Cell isolation and culture

Testes were sterilized in 70% ethanol and transported to the lab on ice Hank’s balanced salt solution (HBSS, Sigma-Aldrich, St. Louis, MO, USA). Collagenase and trypsin digestion followed by filtration were used to isolate NPSC from porcine testes, as described previously³. Three million isolated NPSC were then plated as a single monolayer in tissue culture plates (Falcon, Corning, Inc, Corning, NJ, USA) and cultured overnight at 37°C and 5% CO₂ in 10 mL Dulbecco’s modified eagle medium (DMEM, Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% fetal bovine serum (FBS).

NHS culture and cell collection

The next day, 5 mL of media was removed from each plate and replaced with either 5 mL fresh DMEM or 5 mL of pooled AB human serum with complement preserved (NHS, Innovative Research, Inc., Novi, MI, USA) and plates were incubated for 1.5 h, after which all media was removed, and cells were washed with PBS. NPSC at baseline (NPSC cultured in DMEM only) and NPSC with NHS were lysed with 1 mL Trizole® (Ambion by Life Technologies, Carlsbad, CA, USA) and stored at −80°C. All samples (n = 3 baseline NPSC and n = 3 NHS NPSC) were shipped to GENEWIZ (Azenta Life Sciences, South Plainfield, NJ, USA) in dry ice for RNA isolation and sequencing. GENEWIZ from Azenta Life Sciences then conducted data processing and next generation sequencing, as described previously^8,9. All RNA sequencing data sets related to this study are openly available in the NCBI GEO database (accession number GSE221711).

RNA sequencing

Next generation sequencing was performed by Genewiz from Azenta Life Sciences on the Illumina® NovaSeq^TM or HiSeq® platforms (conditions 2 × 150 base pairs configuration, 20–30 million read depth, data quality of at least 80% bases of at least Q30), which conducted library preparation for RNA including standard and strand specific, small, and ultra-low input RNA. mRNA were the target RNAs for standard and strand-specific analysis including poly(A) selection, mRNA with long noncoding RNA, and ribosomal RNA depletion. Small RNA targets include micro, small interfering, and piwi-interacting RNA selected by size fractionation and adaptor ligation to the 5’ phosphatase. Ultra low-input RNA sequencing used mRNA with poly(A) selection and enriched for full length transcripts. Data set deliverables include quality control reports, aligned data, number of hit counts, differentially expressed genes, gene ontology, gene enrichment analysis, and differential splicing analysis. Further detail on these methods is previously described in Washburn et. al., 2023^8,9.

Discussion

Overall, these data sets include baseline NPSC gene expression, gene expression of NPSC exposed to NHS, and differentially expressed genes of these two data sets. Previously, we have analyzed these data sets for NPSC expression of complement-related genes⁹ and for differential expression of immune-related genes⁸. These data sets can be applied to various other studies involving male reproduction and spermatogenesis, graft protection profiles in xenotransplantation, and protection from autoimmune responses.

Disclosure statement

J.M.D. has stock in Sernova, Corp. Sernova, Corp. has not seen the data or the manuscript. They had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the resuts. R.L.W. declares no conflict of interest.

Additional information

Funding

This research was funded by The CH Foundation (J.M.D.) and the Robert A. Welch Foundation (B1-0028, J.M.D.).