Scrutinizing the human TEX genes in the context of human male infertility
Sieper MH, Gaikwad AS, Fros M, Weber P, Di Persio S, Oud MS, Kliesch S, Neuhaus N, Stallmeyer B, Tüttelmann F, Wyrwoll MJ, 18.08.2023
Abstract
Background: Infertility affects around 15% of all couples worldwide and is increasingly linked to variants in genes specifically expressed in the testis. Well-established causes of male infertility include pathogenic variants in the genes TEX11, TEX14, and TEX15, while few studies have recently reported variants in TEX13B, TEX13C, FAM9A (TEX39A), and FAM9B (TEX39B). Objectives: We aimed at screening for novel potential candidate genes among the human TEX ("testis expressed") genes as well as verifying previously described disease associations in this set of genes. Materials and methods: To this end, we screened the exome sequencing data of 1305 men, including 1056 crypto- and azoospermic individuals, and determined cell-specific expression by analyzing testis-specific single-cell RNA sequencing data for genes with identified variants. To investigate the overarching role in male fertility, we generated testis-specific knockdown (KD) models of all 10 orthologous TEX genes in Drosophila melanogaster. Results: We detected rare potential disease-causing variants in TEX10, TEX13A, TEX13B, TEX13C, TEX13D, ZFAND3 (TEX27), TEX33, FAM9A (TEX39A), and FAM9B (TEX39B), in 28 infertile men, of which 15 men carried variants in TEX10, TEX27, and TEX33. The KD of TEX2, TEX9, TEX10, TEX13, ZFAND3 (TEX27), TEX28, TEX30, NFX1 (TEX42), TEX261, and UTP4 (TEX292) in Drosophila resulted in normal fertility. Discussion: Based on our findings, the autosomal dominant predicted genes TEX10 and ZFAND3 (TEX27) and the autosomal recessive predicted gene TEX33, which all three are conceivably required for germ cell maturation, were identified as novel potential candidate genes for human non-obstructive azoospermia. We additionally identified hemizygous loss-of-function (LoF) variants in TEX13B, TEX13C, and FAM9A (TEX39A) as unlikely monogenic culprits of male infertility as LoF variants were also found in control men. Conclusion: Our findings concerning the X-linked genes TEX13B, TEX13C, and FAM9A (TEX39A) contradict previous reports and will decrease false-positive reports in genetic diagnostics of azoospermic men.
Sieper MH, Gaikwad AS, Fros M, Weber P, Di Persio S, Oud MS, Kliesch S, Neuhaus N, Stallmeyer B, Tüttelmann F, Wyrwoll MJ. Scrutinizing the human TEX genes in the context of human male infertility. Andrology. 2023 Aug 18. doi: 10.1111/andr.13511. Epub ahead of print. PMID: 37594251.
Publication: https://doi.org/10.1111/andr.13511
Disclaimer
The publication Scrutinizing the human TEX genes in the context of human male infertility by Sieper MH, Gaikwad AS, Fros M, Weber P, Di Persio S, Oud MS, Kliesch S, Neuhaus N, Stallmeyer B, Tüttelmann F, Wyrwoll MJ is published under an open access license: http://creativecommons.org/licenses/by-nc/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Curation by the MFGA team Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.
Data set 1: RNA expression profiles of TEX genes
Transcriptome: Single-cell RNA-Sequencing
Species
| Species |
|---|
| Human |
Conditions
| Human phenotype ontology | Participants | Comment |
|---|---|---|
| HP:0000027: Azoospermia | Absence of any measurable level of sperm,whereby spermatozoa cannot be observed even after centrifugation of the semen pellet. | |
| HP:0030974: Cryptozoospermia | A type of oligozoospermia in which spermatozoa can be detected in an ejaculate only after centrifugation and inspection of the pellet. | |
| HP:0012864: Abnormal sperm morphology | A structural anomaly of sperm. | |
| HP:0012207: Reduced sperm motility | An abnormal reduction in the mobility of ejaculated sperm. |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | crypto- or azoospermic | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | 1056 |
| BTO_0001363: testis | terato- or asthenozoospermic | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | 49 |
| BTO_0001363: testis | normozoospermic | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human | 34 |
Images
Figure 1: Single-cell RNA expression of TEX10, ZFAND3 (TEX27), TEX33, FAM9A (TEX39A), and FAM9B (TEX39B), conservation of the amino acid affected by the TEX33 variant and structure of the FAM9 (TEX39) proteins
(A) TEX10 expression is overall weak and shows mild increases in undifferentiated spermatogonia and in spermatocytes during the pachytene stage. (B) ZFAND3 (TEX27) expression is detectable in undifferentiated spermatogonia, during meiosis and during spermiogenesis from the zygotene stage to late spermatids. It is, however, not a germ cell-specific gene, as its expression is also detected in interstitial cells like peritubular myoid cells, Leydig, and Sertoli cells. (C) TEX33 expression is strong in spermatocytes undergoing meiosis and in cells completing spermiogenesis. Its expression is also detectable in interstitial testicular cells. (D) FAM9A (TEX39A) expression was mainly found in spermatocytes during the leptotene stage of prophase I during meiosis. The overall expression is rather weak in testicular cells. (E) FAM9B (TEX39B) expression is weak in undifferentiated spermatogonia, and there is an increase of expression during the first stages of prophase I during meiosis, peaking strongly at the leptotene stage. Expression in somatic cells, if present at all, is extremely weak. (F) Structure of the FAM9 (TEX39) proteins modeled with the Swiss Model shows that FAM9B (TEX39B) exhibits high structural similarity to SYCP3, a component of the synaptonemal complex.
Licensed under: http://creativecommons.org/licenses/by-nc/4.0/
Figure 2: Single-cell RNA expression and predicted protein structure of proteins encoded by the TEX13 gene family
In panels A, C, E, and G, scRNA expression is depicted for the four human TEX13 genes. TEX13B, TEX13C, and TEX13D exhibit extremely low expression in testicular cells. In panels B, D, F, and H, functional domains of the encoded proteins are depicted as determined by Pfam. TEX13A, TEX13C, and TEX13D proteins comprise the N-terminal TEX13 domain as well as the C-terminal Zf-RanBP2 domain. Purple pinheads indicate LoF variants, gray pinheads indicate missense variants identified in infertile men, and white pinheads indicate LoF variants detected in genetically proven fathers.
Licensed under: http://creativecommons.org/licenses/by-nc/4.0/
Data set 2: Fertility assessment of human TEX gene orthologs using Drosophila model organism
Other: Immunofluorescence
Species
| Species |
|---|
| Drosophila melanogaster |
Tissue Types
| BRENDA tissue ontology | Maturity | Description | Species | Replicates |
|---|---|---|---|---|
| BTO_0001363: testis | A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity. | Human |
Images
Figure 3: Fertility assessment and examinations of testes in Drosophila melanogaster with testis-specific KD
At least one ortholog for human TEX2, TEX9, TEX10, TEX13, ZFAND3 (TEX27), TEX28, TEX30, NFX1 (TEX42), TEX26, and UTP4 (TEX292) was available in Drosophila melanogaster. For orthologs of TEX13 and ZFAND3 (TEX27), two different fly constructs were available, respectively. (A) Three rounds of fertility assessment were performed. The mean number of fertilized eggs in all three rounds is indicated on the y-axis. All F1 generation male flies of the RNAi X Nos cross were able to produce offspring in a cross with female WT flies and passed the fertility screening. Error bars represent standard deviation. (B) The diameter of KD and WT fly testes was measured at positions A–D. The diameters did not differ significantly when comparing KD to WT flies. A red asterisk indicates the apex. (C) Whole testis length was measured in KD and WT flies. No significant differences were observed in testis length when comparing KD to WT flies. A red asterisk indicates the apex. (D) Testicular structure was investigated using immunofluorescence (IF) staining with DAPI (DNA staining; blue) and phalloidin (F-actin staining; purple). As an example, IF staining in WT and TEX2 KD testis is shown. No differences in testicular structures were observed when comparing KD to WT fly testes.
Licensed under: http://creativecommons.org/licenses/by-nc/4.0/