The germ cell-specific proteome
Germ cells give rise to the gametes in humans, which are cells that can undergo meiosis, as opposed to somatic cells that undergo mitosis. Gametes are diploid germ cells that undergo development into haploid eggs and sperm through oogenesis and spermatogenesis.
Transcriptome analysis shows that 82% (n=16552) of all human proteins (n=20162) are detected in germ cells and 5400 of these genes show an elevated expression in any germ cells compared to other cell type groups. In-depth analysis of the elevated genes in germ cells using scRNA-seq and antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in the following types of germ cells: spermatogonia, spermatocytes, early spermatids and late spermatids in testis, and oocytes in the ovaries.
The germ cell transcriptome
The scRNA-seq-based germ cell transcriptome can be analyzed with regard to specificity, illustrating the number of genes with elevated expression in each specific germ cell type compared to other cell types (Table 1). Genes with an elevated expression are divided into three subcategories:
As shown in Table 1, 528 genes are elevated in spermatogonia compared to other cell types. Spermatogonia are diploid cells that form the basal layer of the seminiferous duct and present the initial phase of the spermatogenesis, before meiosis. The spermatogonia undergo asymmetric cell division resulting in two subtypes, type A cells that have stem cell-like properties and maintain the spermatogonia population, and type B spermatogonia that will continue to evolve into primary spermatocytes. Examples of genes expressed in premeiotic spermatogonial cells include testis-specific protein, Y-linked 2 (TSPY2), a transcription factor primarily expressed in the nuclei of spermatogonia, which plays a key role in male sex determination and differentiation by controlling testis development and male germ cell proliferation. Another example is sarcoma antigen 1 (SAGE1), which is a cancer related gene that belongs to the Cancer Testis Antigen (CTA) family, suggested to constitute important targets for immunotherapy.
As shown in Table 1, 1415 genes are elevated in spermatocytes compared to other cell types. Spermatocytes are derived from type B spermatogonia and can be subdivided into primary spermatocytes that enter the first meiosis, and secondary spermatocytes that enter the second meiosis to produce haploid spermatids. The longest phase of meiosis is prophase I which is subdivided into different stages including preleptotene and pachytene. Several of the testis-specific proteins localized to spermatocytes are involved in testicular differentiation, proliferation, and meiosis. Pachytene spermatocytes are easily recognizable by being the only meiotic germ cells observed on testicular histology sections due to the long duration of meiotic prophase I. The structural synaptonemal complex protein (SYCP3) is involved in the recombination and segregation of meiotic chromosomes. The synaptogyrin 4 (SYNGR4) protein is a membranous protein that belongs to the synaptogyrin family and its function is unclear.
As shown in Table 1, 3053 genes are elevated in early spermatids compared to other cell types. Spermatids result from the division of secondary spermatocytes and hence meiosis completion. These cells are divided into early spermatids (round spermatids) and late spermatids (elongated spermatids). Although spermatids are not divided, they undergo a complex process of metamorphosis (called spermiogenesis). Early spermatids are transcriptionally active. One example of an early spermatid protein is the sperm acrosome associated 4 (SPACA4), which is a sperm surface membrane protein that may be involved in sperm-egg plasma membrane adhesion and fusion during fertilization. Another example is the protein product of actin like 7B (ACTL7B), which is expressed only in testis where its exact function is uncertain but possible functions could be nuclear migration and chromatin remodeling.
As shown in Table 1, 2978 genes are elevated in late spermatids compared to other cell types. The late spermatid will eventually mature into spermatozoa which will be released in the seminiferous tubule lumen. The DNA will become densely packed and form an acrosome and a mid area with mitochondria. Different from the early spermatids, late spermatids are transcriptionally inert when the acrosome is fully developed because of the tightly packed chromatin. Examples of cell type enriched proteins in late spermatids are spermatogenesis associated 3 (SPATA3) and protein phosphatase 1 regulatory subunit 32 (PPP1R32). However, their function is yet to be studied.
The female germ cells, oocytes, are produced in the ovaries, in an anatomical structure called the ovarian follicle. Oogenesis is a discontinuous process that starts during fetal life with the development of the primary oocyte. A single layer of granulosa cells surrounds each primary oocyte, arrested in prophase I until puberty. The granulosa cells, in turn, are enclosed in a thin layer of extracellular matrix, called the zona pellucida. These structures are referred to as primordial follicles. At puberty, the primordial follicles eventually develop into primary, secondary, and tertiary vesicular follicles. Each month, typically only one developing primary follicle becomes dominant and achieves complete maturation to release the oocyte into the fallopian tube. Examples of cell type enriched proteins in oocytes include folliculogenesis specific bHLH transcription factor (FIGLA) and zona pellucida glycoprotein 3 (ZP3). FIGLA is a transcription factor that plays an important role in the regulation of oocyte-specific genes essential for normal ovarian follicular development, including ZP3, a component of the zona pellucida, a structure that is important for subsequent fertilization by a sperm cell.
Germ cell function
Germ cells are precursor cells to the gametes of the human male and female. These cells are the only cells in the body that undergo meiosis instead of mitosis as compared to other somatic cells. The germ cells will differentiate into egg and sperm cells through spermatogenesis (sperm cell development) and oogenesis (egg cell development). When germ cells differentiate into unfertilized eggs and sperm they will become haploid (half of the genetic code) gametes through meiosis. Haploid cells, when merged, will contribute to genetic differences in the offspring, creating individuals that are genetically different.
The histology of organs that contain germ cells, including interactive images, is described in the Protein Atlas Histology Dictionary.
Here, the protein-coding genes expressed in germ cells are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in different germ cell types.
The transcript profiling was based on publicly available genome-wide expression data from scRNA-seq experiments covering 29 tissues and peripheral blood mononuclear cells (PBMCs). All datasets (unfiltered read counts of cells) were clustered separately using louvain clustering, resulting in a total of 557 different cell type clusters. The clusters were then manually annotated based on a survey of known tissue and cell type-specific markers. The scRNA-seq data from each cluster of cells was aggregated to mean normalized protein-coding transcripts per million (nTPM) and the normalized expression value (nTPM) across all protein-coding genes. A specificity and distribution classification was performed to determine the number of genes elevated in these single cell types, and the number of genes detected in one, several or all cell types, respectively.
It should be noted that since the analysis was limited to datasets from 29 tissues and PBMC only, not all human cell types are represented. Furthermore, some cell types are present only in low amounts, or identified only in mixed cell clusters, which may affect the results and bias the cell type specificity.
Relevant links and publications
Uhlén M et al., Tissue-based map of the human proteome. Science (2015)