This study assessed the feasibility of testis tissue cryopreservation (TTC) for fertility preservation in prepubescent boys with cryptorchidism. From January 2014 to December 2022, the University Hospital of Copenhagen (Rigshospitalet, Copenhagen, Denmark) implemented TTC for 56 boys with cryptorchidism to preserve their reproductive potential. Testis tissue samples were collected during orchiopexy (32 cases) or at subsequent follow-up procedures (24 cases), necessitated by an increased risk of infertility as indicated by hormonal assessments and/or findings from initial surgical biopsies. Testis samples were procured for TTC and pathological analysis. The cohort had an average age of 1.3 (range: 0.3-3.8) years at the time of orchiopexy, with 91.1% presenting bilateral cryptorchidism. The study revealed a median germ cell count of 0.39 (range: 0-2.88) per seminiferous tubule, with germ cells detected in 98.0% of the bilateral biopsies and 100% of the unilateral, indicating a substantial potential for fertility in these immature tissues. A dark spermatogonia (Ad) was detected in 37 out of 56 patients evaluated, with a median Ad spermatogonia count of 0.027 (range: 0.002-0.158) per seminiferous tubule. A total of 30.2% of the samples lacked Ad spermatogonia, indicative of potential gonadotrophin insufficiency. The median hormone levels measured were as follows: follicle-stimulating hormone (FSH) at 0.69 (range: 0.16-2.5) U l -1 , luteinizing hormone (LH) at 0.21 (range: 0.05-3.86) U l -1 , and inhibin B at 126 (range: 17-300) pg ml -1 . Despite early orchiopexy, 20%-25% of boys with cryptorchidism remain at risk for future infertility, substantiating the necessity of TTC as a precaution. The study highlights the need for refined predictive techniques to identify boys at higher risk of future infertility.
Humans ; Male ; Cryptorchidism/pathology* ; Cryopreservation ; Testis/pathology* ; Fertility Preservation/methods* ; Child, Preschool ; Infant ; Orchiopexy ; Spermatogonia/pathology* ; Infertility, Male/etiology*

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.
Animals ; Atrophy ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoproteins ; metabolism ; physiology ; Homeostasis ; Isoenzymes ; metabolism ; Male ; Mice ; Nerve Tissue Proteins ; metabolism ; physiology ; Phosphoglycerate Kinase ; metabolism ; Polypyrimidine Tract-Binding Protein ; metabolism ; physiology ; RNA, Messenger ; metabolism ; RNA-Binding Proteins ; Seminiferous Tubules ; pathology ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; metabolism ; Testis ; metabolism

OBJECTIVE: To determine the molecular mechanism of germ cell apoptosis via investigating the effect of PFT-α on the expression of p53 and bcl-2/bax during experimental cryptorchid cell apoptosis. METHODS: Male Sprague-Dawley rats were assigned into 4 groups: a sham-operated group, a cryptorchid group, a cryptorchid+p53 inhibitor (p53 inhibitor-alpha, PFT-α) group, and a cryptorchid+dissolvent of PFT-α [dimethyl sulphoxide (DMSO)] group. Unilateral cryptorchidism was surgically induced in the rats of the cryptorchid group, PFT-α group, and cryptorchid+dissolvent of PFT-α group. The rats in the PFT-α group and cryptorchid+dissolvent of PFT-α group were intra-peritoneally injected PFT-α and dissolvent of PFT-α, respectively, once a day. The rats were killed on the 7th day after the surgery. The morphology of spermatogenic epithelium at the side of surgery in the rats was observed under light microscope. The apoptosis of spermatogenic cells in the unilateral cryptorchidism was evaluated by TUNEL and flow cytometry (FCM). The protein expression levels of p53, bcl-2, and Bax were detected by Western blot and immunohistochemical assay in turn. RESULTS: Compared with the cryptorchid groups and the cryptorchid+dissolvent of PFT-α group, the seminiferous epithelium of the cryptorchid+p53 inhibitor group appeared orderly, with thicker cell layers and lower apoptosis index, weak protein expression level of p53/Bax and strong protein expression level of bcl-2. CONCLUSION PFT-α inhibits the germ cell apoptosis caused by the experimental cryptorchidism via increasing the expression of bcl-2 and decreasing the expression of p53 and bax.
Animals ; Apoptosis ; Benzothiazoles ; pharmacology ; Cryptorchidism ; pathology ; Disease Models, Animal ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; drug effects ; Toluene ; analogs & derivatives ; pharmacology

Animals ; Apoptosis ; Male ; Morphine Dependence ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; metabolism ; pathology ; Spermatozoa ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

AIMTo investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.

METHODSMice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.

RESULTSLow doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.

CONCLUSIONE2 has a dose-related mitogenic effect on spermatogonia.

Animals ; Cell Division ; drug effects ; Cryptorchidism ; physiopathology ; Disease Models, Animal ; Estradiol ; blood ; pharmacology ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Mice ; Spermatogonia ; cytology ; drug effects ; pathology ; Testosterone ; blood

OBJECTIVETo evaluate the role of spermatic nerves in the regulation of spermatogenesis.

METHODSFifty-four mature SD male rats (350-375 g) were randomized into a sham operation group (SO) and three experiment groups, and the latter underwent bilateral surgical removal of the superior spermatic nerve (SSN) or/and the inferior spermatic nerve (ISN). The animals were killed 1 month and 2 months after the operation. HE stain was used to observe spermatogenesis. Transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) were employed to detect apoptosis.

RESULTSImpaired spermatogenesis was observed 2 months after the operation, with only Sertoli cells and a few spermatogonia remaining in the regressed tubules in all the treatment groups. The abnormal tubules in the SSN, ISN and SSN + ISN denervated testes accounted for (13.25 +/- 2.03)%, (11.0 +/- 4.36)% and (34.17 +/- 3.78)% respectively. Chromosome condensation and fragmentation in the germ cells were observed under the electron transmission microscope in all the denervated testes. TUNEL showed the spermatogonia and Leydig cells to be apoptotic in all the denervated testes and the incidence of the apoptotic cells in the SSN + ISN denervated testes was significantly higher than in the SSN or ISN denervated ones.

CONCLUSIONSpermatic nerves play an important role in spermatogenesis.

Animals ; Apoptosis ; Denervation ; Germ Cells ; pathology ; Leydig Cells ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatic Cord ; innervation ; Spermatogenesis ; physiology ; Spermatogonia ; pathology ; Testis ; innervation

OBJECTIVESTo study the effect of testicular local heating on spermatogenic cell apoptosis in rat.

METHODSSeventy male SD rats were divided into heat treatment group (43 degrees C) and control group (22 degrees C). Each group was further divided into seven sub-groups respectively according to the time of 12 hours and 1 days, 3 days, 6 days, 10 days, 50 days and 80 days after testicular local treatment. The spermatogenic cell apoptosis in all sub-groups was examined by means of electron microscopy, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUDP-nick end labeling(TUNEL) method.

RESULTSIn the groups of heat treatment, spermatogenic cell apoptosis was detected by electron microscopy; flow cytometry showed that the percentage of cells with sub-haploid increased(P < 0.01); the percentage of positive TUNEL cells in the heat treatment groups was higher than that in the control group(P < 0.01). Initiation of spermatogenic cell apoptosis after testicular heating was not random but was highly selective.

CONCLUSIONSLocal testicular heating could increase the spermatogenic cell apoptosis. The most sensitive cell is spermatocyte. Spermatid and sperm also display apparent changes. Heating can increase the apoptosis of spermatogonia in a long period.

Animals ; Apoptosis ; Flow Cytometry ; Hot Temperature ; In Situ Nick-End Labeling ; Male ; Microscopy, Electron ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; ultrastructure ; Testis ; pathology ; ultrastructure

PURPOSE: Although retractile testes are frequently found in the pediatric population, there are controversies in the management of retractile testes. We investigated the necessity of treatment for retractile testes by analyzing their histologic findings. MATERIALS AND METHODS: Sixty-one testicular biopsies were performed during orchiopexy from 36 boys(range: 1.3-12.9 years, mean: 5.4 years) with retractile testes(11 unilateral, 50 bilateral) and 115 testicular biopsies from 83 cryptorchid patients(range: 0.6-15.0 years, mean: 3.7 years, 51 unilateral, 64 bilateral). Parameters for both Sertoli cell and germ cell were determined in each group. RESULTS: The average tubular degeneration phase(TDP) V-VII were 0.23+/-0.18 for retractile testes and 0.22+/-0.17 for cryptorchid testes and were not statistically different. Both the average sertoli cell index(SCI) and mean spermatogonia per tubules(S/T) value were statistically different between retractile and cryptrochid testes with values of 26.81+/-6.75, 23.04+/-5.85(p<0.01) and 2.96+/-1.33, 0.61+/-0.87(p<0.01), respectively. CONCLUSIONS: Although S/T value of retractile testes was higher than that of cryptorchid testes, Sertoli cell degenerative patterns were similar. These findings might indicate that retractile testis needs treatment like cryptorchid testis does. However, further investigation is warranted to elucidate whether these changes are normal variations since changes are observed in both Sertoli & germ cells in normal boys as they are aging.
Aging ; Biopsy ; Germ Cells ; Orchiopexy ; Pathology ; Spermatogonia ; Testis*