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Federal government websites often end in. The site is secure. Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients.

Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells SSCs of cryptorchidism patients by retinoic acid RA and stem cell factor SCF treatment. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells.

Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients. Male gametogenesis is a process by which spermatogonial stem cells SSCs divide and differentiate into haploid spermatids.

Any error during male gametogenesis can result in male infertility, which is a major health problem around the world De Kretser and Baker, Cryptorchidism is one of the most common causes that result in NOA Sinnar et al. Severe cryptorchidism could lead to male infertility, since male germ cells especially haploid spermatids are significantly reduced or completely lost in cryptorchid testes Zivkovic et al. It has been reported that the transition of gonocytes into A dark spermatogonia in cryptorchid testes is impaired Kamisawa et al.

Therefore, it is of great significance to establish an effective method to induce differentiation of human spermatogonia from cryptorchid testes into haploid spermatids for the treatment of male infertility. Previous studies have been focused on the in vitro models of male germ cell maturation Tesarik, However, there is currently no efficient approach for generating haploid spermatids in vitro from spermatogonia of human testes.

Complete spermatogenesis in vitro to obtain male gametes has not yet been achieved in humans, although certain progress has been made in the derivation of male germ cells from mouse or human embryonic stem cells ESCs Aflatoonian et al.

There are ethical issues obtaining human ESCs, which is a major obstacle for their potential use in the clinic. It has recently been demonstrated that the induced pluripotent stem cells iPSCs could generate primordial germ cells and finally haploid spermatids Easley et al. Of great concern, male germ cells derived from human iPSCs may not be used for treating male infertility due to tumor-forming risks, which result from the reprogramming of somatic cells by gene transfer using viral vectors and their genetic instability.

Therefore, more attention has been paid to generating male gametes from human spermatogonia of patients. It has been suggested that several growth factors, such as bone morphogenetic proteins BMPs , glia cell line-derived neurotrophic factor GDNF , stem cell factor SCF , and retinoic acid RA , were crucial for the maintenance of normal spermatogenesis in rodents.

Furthermore, SCF is required for the proliferation of mouse differentiating spermatogonia, specifically type A 1 to A 4 spermatogonia Hasthorpe, ; Tajima et al. RA, the active derivative of vitamin A, controls the entry of germ cells into meiosis in both mice and humans Childs et al. Therefore, RA and SCF were chosen in this study to induce the differentiation of human spermatogonia from cryptorchid testes.

It has been recently reported by our peers and us that human SSCs can be clearly identified and cultured for a short- and long-term period He et al. Round spermatids with unknown function can be derived from mouse spermatogonia Feng et al. Nevertheless, the generation of functional haploid spermatids from SSCs in vitro has not yet been achieved in humans. Here, we present molecular and cellular evidence demonstrating the differentiation of human SSCs from cryptorchid patient into cells with phenotypic characteristics, DNA content, and fertilization and development capacity of haploid spermatids.

Of unusual significance, our ability to generate human functional haploid spermatids from cryptorchid testes could offer an important source of functional and autologous male gametes for treating male infertility in azoospermia patients. We first checked the chromosome karyotype and the expression of numerous Y chromosome genes of cryptorchid patients.

Karyotype analysis revealed that cryptorchid patients possessed a normal chromosome karyotype Figure 1 A. Multiplex PCR was used to check whether cryptorchid patients had Y chromosome microdeletion. As shown in Figure 1 C, numerous Y chromosome genes, including SRY , sY , sY , sY86 , sY , sY84 , and sY , were detected in cryptorchid patients, which was comparable to the expression of these genes in normal men Figure 1 B , suggesting that cryptorchid patients did not have Y chromosome microdeletion.

Therefore, testicular tissues of these cryptorchid patients were used to induce differentiation. The clinic data of cryptorchid patients are shown in Table S1 available online.

The levels of testosterone T and estradiol E2 of cryptorchid patients were within the normal ranges. However, both left and right testicular volumes of cryptorchid patients were significantly smaller than those of normal men.

The levels of follicle-stimulating hormone FSH , luteinizing hormone LH , and prolactin PRL in cryptorchid patients were statistically higher than those of normal men. Histological and immunohistochemical analyses of cryptorchid patients were performed to evaluate the spermatogenesis status of testicular tissues.

The testes from obstructive azoospermia OA patients due to inflammation but with normal spermatogenesis in vivo served as the controls. Histological examination showed that seminiferous tubules of cryptorchid testes had a reduced tubular diameter and a thickened basement membrane Figure 2 B compared to the control testes Figure 2 A. There were human spermatogonia along the basement membrane in cryptorchid testes Figure 2 B ; however, differentiated male germ cells, including spermatocytes and haploid spermatids, were very rare or completely lost in the seminiferous tubules of cryptorchid testes Figure 2 B compared with the control testes Figure 2 A.

To verify specific staining of MAGEA4, replacement of primary antibody with normal mouse immunoglobulin G IgG was used as a negative control, and no positive reaction was seen in normal testes Figure 2 E or the testes of cryptorchid patients Figure 2 F. The seminiferous tubules of cryptorchid testis were also characterized by immunostaining with SCP3 synaptonemal complex protein 3 , a specific marker for meiotic germ cells West et al.

Immunohistochemistry showed that very few cells were positive for SCP3 in the seminiferous tubules of cryptorchid testis Figure 2 H compared to the control testes Figure 2 G. Replacement of primary antibody with normal rabbit IgG and PBS served as a negative control, and no staining was observed in normal testes Figure 2 I and data not shown or the testes of cryptorchid patients Figure 2 J and data not shown , which confirmed the specific staining of SCP3.

Taken together, these results reflect that spermatogonia remained whereas meiotic male germ cells were rare or lost in the testes of cryptorchid patients. Human male germ cells were isolated from testis tissues of 16 cryptorchid patients and 9 OA patients using two-step enzymatic digestion followed by differential plating Figure 3 A. Replacement of primary antibodies with PBS or normal IgG served as a negative control, and no staining was observed data not shown.

Collectively, these data suggest that the freshly isolated male germ cells were phenotypically human SSCs. The expression of SYCP1 1. RA and SCF have been reported to play important roles in promoting spermatogenesis in rodents, and thus they were utilized to induce the differentiation of human SSCs from cryptorchid patients.

The cells in these colonies were human SSCs with proliferation potential He et al. We next evaluated the differentiation potential of human SSCs from cryptorchid patients. These results suggest that RA and SCF induce the differentiation of human SSCs from cryptorchid patients and OA patients into meiotic male germ cells and haploid cells at transcriptional levels.

Protamine 2 and acrosin are generally regarded as markers for haploid cells. D Immunocytochemistry showing acrosin expression in the sorted cells of the differentiated cells from cryptorchid patients upper panel.

The expression of acrosin in donated normal human sperm served as positive controls lower panel. Notably, haploid cells were increased from 7. Round spermatid microinjection ROSI was performed to determine the fertilization capacity of human round spermatids generated from human SSCs of cryptorchid patients and OA patients. A—D Phase-contrast microscope showing the morphology of embryos with two pronuclei PN A , embryos developing into the two-cell B , four-cell C , and eight-cell D stages.

E Immunocytochemistry revealing the expression of HumNuc in the embryos from mouse oocytes fertilized with human round spermatids from human SSCs. H Clustering of the transcriptome of single embryo derived from round spermatids of cryptorchid patients and from the nucleus of Sertoli cells by single-cell RNA sequencing analysis.

To verify the fertilization of human round spermatids generated from human SSCs of cryptorchid patients with mouse oocytes and exclude parthenogenetic activation of mouse oocytes themselves, immunocytochemistry was carried out using human antibody against HumNuc. The embryos derived from mouse oocytes fertilized with human round spermatids of cryptorchid patients were positive for human anti-HumNuc Figure 6 E. Replacement of human anti-HumNuc with normal rabbit IgG Figure 6 F or PBS Figure 6 G in the embryos developed from mouse oocytes fertilized with human round spermatids served as a negative control, and no staining was seen.

Moreover, single-cell RNA sequencing analysis revealed that there were 26,, total reads and 18,, total mapped reads in the embryos derived from round spermatids, while 24,, total reads and 13,, total mapped reads were detected in the embryos generated from the nucleus of Sertoli cells. We identified 9, genes in the embryos derived from round spermatids and the nucleus of Sertoli cells Figure S5 A , and there were 5, differentially expressed genes DEGs Figure 6 H; Figure S5 B and distinct distribution of gene coverage Figure S5 C between the embryos from round spermatids and the nucleus of Sertoli cells.

To further observe the dynamic changes during embryo development, H3K9 trimethylation H3K9-TriM was applied to label the maternal genomes. As shown in Figure S6 , maternal pronuclei were positive for H3K9-TriM, while its staining in male pronucleus was hardly detectable in each group.

Sperm chromatin began to decondense immediately after fertilization Figures S6 A and S6B , and male and female pronuclei formed at 4 hr and 6 hr after fertilization Figures S6 C and S6D. Interestingly, the embryos derived from round spermatids showed a different distribution pattern of H3K9-TriM compared to embryos derived from PBS Figure S6 F or from the nucleus of Sertoli cells with both pseudopronuclei and female pronuclei Figure S6 G.

Collectively, these results clearly implicate that human round spermatids derived from human SSCs of cryptorchid patients have both fertilization and development potential. We highlight the generation of human functional haploid spermatids from human SSCs of cryptorchid patients.

Cryptorchidism is the most common etiologic factor for azoospermia in adults. However, the exact causes of most cases of cryptorchidism remain unknown Philibert et al. It has been suggested that a reduced total number of male germ cells in cryptorchid testes is the cause of male infertility Hadziselimovic and Herzog, Therefore, in vitro differentiation of human SSCs into haploid spermatids from cryptorchid testes could be an ideal method for treating infertility in cryptorchid patients.

Here, we have shown obvious evidence for rescuing germ cell development in cryptorchid patients using in vitro techniques, as evidenced by our finding that human SSCs from cryptorchid patients can progressively differentiate into meiotic and haploid spermatids by treatment with RA and SCF.

Notably, human spermatogonia exist whereas meiotic male germ cells are rather rare or completely lost in the testis of cryptorchid patients, since there were numerous cells positive for MAGEA4, a marker for human spermatogonia He et al. This conclusion can also be verified by our observations that almost all freshly isolated male germ cells from cryptorchid patients were positive for UCHL1, a marker for human spermatogonia He et al.

Our results are consistent with previous findings showing defective maturation of germ cells in cryptorchid patients Agoulnik et al. Therefore, testis tissues of these cryptorchid patients were chosen for differentiating into spermatocytes and haploid spermatids. The starting cells we used for differentiation were colony cells. We found that isolated cells from cryptorchid testes were able to proliferate and form colonies composed of numerous cells.

We and others have revealed that the cells in these colonies were SSCs with proliferation potential He et al. It has been demonstrated by xenotransplantation that the cells in the colonies were actually human SSCs with self-renewal capacity Sadri-Ardekani et al. SCP3 can be used to measure the synaptonemal complex, while CREST is a hallmark for detecting centromeric regions and MLH1 has been utilized for measuring meiotic recombination frequency Holloway et al.

Our results, using these markers for meiosis and postmeiosis, clearly indicate that RA and SCF could induce human spermatogonia to enter the postmeiotic stage and eventually differentiate into haploid spermatids.

We have previously reported that RA can act as a meiosis-inducing factor in the differentiation of iPSCs into male germ cells Yang et al. SCF has been shown to be essential in spermatogonial differentiation as well as meiotic initiation Feng et al.

Furthermore, the crosstalk between RA and the SCF pathway could stimulate differentiation of male germ cells toward the meiotic stages Pellegrini et al. Consistent with these findings, we found that RA and SCF could efficiently induce the differentiation of human SSCs from cryptorchid testes into postmeiotic male germ cells.

Taken together, these studies from our peers and us suggest that RA and SCF are effective to coax the second meiosis of germ cells into haploid spermatids.

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