Human Reproduction · 2002 · 275 citations · open access
Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination
AbstractBACKGROUND: Fertility protection is an urgent clinical problem for prepubertal male oncology patients who undergo either chemotherapy or radiotherapy. As these patients do not have mature sperm to be frozen, there is as yet no effective method to preserve their fertility. METHODS AND RESULTS: Single pieces of immature mouse (1.5 x 1.5 x 1.5 mm) or rabbit (2.0 x 2.0 x approximately 3.0 mm) testis were cryopreserved, thawed and transplanted into mouse testes. Histological techniques were used to determine the presence of spermatogenesis, which was restored in both mouse and rabbit testicular pieces, and led to the production of mature sperm after both cryopreservation and syngeneic or xenogeneic transplantation into mouse testes. Using sperm developed in the frozen-thawed transplants, mouse offspring were born after in-vitro microinsemination. Furthermore, rabbit offspring were obtained using rabbit sperm that developed in fresh transplants in a xenogeneic surrogate mouse. CONCLUSIONS: This approach of 'testicular tissue banking' is a promising technique for the preservation of fertility in prepubertal male oncology patients. Xenogeneic transplantation into immunodeficient mice may provide a system for studying spermatogenic failure in infertile men.
https://doi.org/10.1093/humrep/17.12.3039Clinical and Translational Medicine · 2023 · 8 citations · open access
A pathogenic <i>AKAP4</i> variant, p.R429H, causes male in/subfertility in humans and mice
AbstractDear Editor, Azoospermia, oligozoospermia and asthenozoospermia are well-established causes of male infertility. Next-generation sequencing has contributed to understanding Mendelian forms of male sterility.1 We identified a pathogenic hemizygous AKAP4 variant (c.1286G > A/p.R429H) shared by two siblings suffering from non-obstructive azoospermia (NOA), while a different missense change involving the same amino acid residue, p.R429C, caused multiple morphological abnormalities of the sperm flagellum (MMAF) and severe oligozoospermia in a prior study.2 An equivalent Akap4R428H mutation knock-in mouse model was generated using CRISPR/Cas9 technology, which exhibited pronounced male subfertility characterized by diminished sperm count and motility, as well as fibrous sheath (FS) abnormalities in the flagella. A-kinase anchor protein 4 (AKAP4), an X chromosome-linked gene, is exclusively expressed in spermatids and mature spermatozoa in previous studies.3 AKAP4 participates in tethering Cyclic-AMP dependent protein kinase A (PKA) to substrates for protein phosphorylation and constructing FS skeleton structure.4 Recently, testicular single-cell transcriptomic studies5, 6 have indicated that AKAP4, which escapes meiotic sex chromosome inactivation, is also expressed in spermatogonia and spermatocytes, despite lower expression than in spermatids. This knowledge suggests that AKAP4 might function beyond flagella development during spermatogenesis. Notably, variants in the AKAP4 gene have been identified in infertile males exhibiting distinct phenotypes, such as asthenozoospermia,7 MMAF,2 and azoospermia/NOA,8, 9 which shed light on the divergence of male infertility phenotypes of AKAP4 mutations. We recruited a family including two brothers with histopathologically confirmed NOA (Figure 1A,B). The characteristics of the two probands (II-1, II-2) are shown in Table 1. After precluding common etiological factors, whole-exome sequencing was applied to search for genetic causes (Figure 1C). After strictly filtering, six genes remained: three showed biallelic missense mutations (OBSCN, SYNE1 and ZNF282), and three had X-linked missense mutations (AKAP4, PLXNB3, and SRPK3) (Table S1, Figure S1). The contributions of mutations in OBSCN, SYNE1, PLXNB3 and SRPK3 to NOA phenomena were excluded by previous knockout (KO) mouse studies through a search of the Mouse Genome Informatics database (https://www.informatics.jax.org/). Segregation analysis was applied to identify hemizygous AKAP4 variation (Figure 1D, Table S2) and compound heterozygous ZNF282 variations (Figure S2). AKAP4 c.1286G > A/p.R429H is located in exon 5, and in silico information is detailed in Table 1 and Figure 1E–G. As yet, there is no literature report of Akap4 homologous mutant knock-in and Zfp282-KO mouse models (mouse ZFP282 is orthologous to human ZNF282), so we constructed corresponding mice to explore the effect of these variants on male fertility. AKAP4 variant Zfp282-KO mice (Figure S3A–C, Table S3 and S4) were viable and exhibited no overt abnormalities. A series of experiments were applied to test the fertility of Zfp282-KO mice, including fertility test (Figure S3D), sperm counts and motility (Figure S3E), H&E staining of testis/epididymis sections and Papanicolaou staining of sperm (Figure S3F), which collectively showed that there is no impact of ZNF282 on male fertility. We generated Akap4 p.R428H mice (Figure 2A,B, Table S5 and S6), which is equivalent to the human AKAP4R429H mutation. The AKAP4 protein was significantly reduced in the testes of Akap4R428H mice (Figure 2C), indicating that the R428H variant might affect AKAP4 protein stability. The fertility test indicated severe male subfertility in which only 8/24 female mice mated with Akap4R428H males were pregnant and produced 37 offspring; in contrast, 24/25 female mice mated with wild-type (WT) males became pregnant and gave rise to 203 offspring (Figure 2D). The male reproductive system, testis/body weight ratio and histological examination of testis sections of Akap4R428H mice were not obviously different from those of WT mice (Figure 2E–F). Transmission electron microscopy analysis showed no obvious abnormalities in manchette structure of spermatids in Akap4R428H mice (Figure S4). To illustrate the cause of reduced fertility in Akap4R428H mice, we performed sperm analysis using mature sperm from the cauda epididymis. Significantly reduced sperm count and progressive motility were identified in Akap4R428H mice (Figure 2G). Papanicolaou staining further indicated that Akap4R428H mice produced short-tailed and bent-tailed sperm with dramatically attenuated principal piece (Figure 2H). Short-tailed and bent-tailed sperm accounted for approximately 33% and 25% of the total sperm in Akap4R428H, respectively (Figure 2I). TEM showed that FS was either unrecognizable or partially lost (Figure 2J). The ratio of FS deficiency in sperm from Akap4R428H was significantly higher than that in WT sperm (54.00% vs. 5.90%) (Figure 2K). We further found that the protein expression of AKAP3 (a FS protein) and QRICH2 (a known target of AKAP4) was significantly lower in the testis lysates of Akap4R428H mice (Figure 2L,M). Collectively, these data confirmed that the R429H variant of AKAP4 is a pathogenic mutation to cause male in/subfertility. To exploit the regulatory mechanism of AKAP4 in mouse spermatogenesis, we reanalyzed the single-cell transcriptome data of Akap4-KO and WT testes from the Sequence Read Archive database (access number: SRR9107534),10 which were detailed in the supplementary Material and Methods (Figure S5A,B). Through differential expression analysis without distinguishing cell types we identified thirteen main differentially expressed genes (DEGs) (|log2FC|≥.5, adjusted p < .05) including Ccdc38 and Haspin, which were reported in previous literature10 (Figure S5C). Trajectory analysis provided novel findings compared with the previous results.10 The shorter velocity vectors indicated a decreased accumulation of mRNA in Akap4-KO mice (Figure S6A). Round spermatids (RSs) were positioned at the starting point of the pseudotime trajectory in KO mice, whereas spermatocytes served as the early state of cell differentiation in WT mice (Figure S6B,C). Enrichment of RS DEGs did not show a significant pathway for spermatogenesis (Figure S7A–C). Protein-protein interaction network (PPI) analysis of these DEGs showed potential AKAP4 interacting partners (Figure S7D,E). Both up/downregulated DEGs in elongating spermatids were significantly enriched in spermatogenesis (Figure S6D,E), and PPI revealed a central node containing AKAP4, H1FNT (Figure S6F,G), suggesting that AKAP4 may act through its interactors to mainly affect the late stages of spermiogenesis. In conclusion, the functional alterations in AKAP4 have been demonstrated to exert significant contributions to male infertility, encompassing asthenozoospermia (including MMAF), severe oligozoospermia and even a complete failure of spermatogenesis (Figure 3). The underlying mechanisms of AKAP4 mutations leading to NOA and the phenotypic difference between the AKAP4 p.R429H variant in humans and Akap4R428H mice remain to be studied in the future. Wei H and Zhang XH performed the major experiments and wrote the manuscript. Wang CY. and Wang J undertook the bioinformatics analysis. Li TY completed Sanger sequencing. Chen SR, Li HJ and Wang BB designed the study and revised the manuscript. All authors approved the final version for submission. The authors thank all of their colleagues for technical support. The authors declare no competing interests in relation to publication of this study. This work was supported by the Beijing Municipal Natural Science Foundation (7232112), the National Key Research and Development Project (2019YFA0802101) and the Open Fund of Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education. This study was approved by the ethics committee from the Peking Union Medical College Hospital and National Research Institute for Family Planning. Animal experiments were approved by the Animal Care and Use Committee of the College of Life Sciences, Beijing Normal University. The datasets generated during the current study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
https://doi.org/10.1002/ctm2.1463