Surgery-induced cryptorchidism induces apoptosis and autophagy of spermatogenic cells in mice.

SummaryCryptorchidism, characterized by the presence of one (unilateral) or both (bilateral) undescended testes, is a common male urogenital defect. Cryptorchidism can lead to male infertility, testicular cancer being the most extreme clinical symptom, as well as psychological issues of the inflicted individual. Despite this, both knowledge about the aetiology of cryptorchidism and the mechanism for cryptorchidism-induced male infertility remain limited. In this present study, by using an artificial cryptorchid mouse model, we investigated the effects of surgery-induced cryptorchidism on spermatogenic cells and seminiferous epithelial cycles. We found that surgery-induced cryptorchidism led to a reduced testicular weight, aberrant seminiferous epithelial cycles and impaired spermatogenesis characterized by degenerating spermatogenic cells. We also observed multinucleated giant cells after surgery-induced cryptorchidism. Transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot assays demonstrated cryptorchidism-induced apoptosis of spermatogenic cells. Moreover, we identified the occurrence of autophagy in germ cells after surgery-induced cryptorchidism. Interestingly, apoptosis and autophagy were synchronous, suggestive of their synergetic roles in promoting germ cell death. Our results provide novel insights into the cryptorchidism-induced male infertility, thereby contributing to the development of male contraceptive strategies as well as treatment options for male infertility caused by cryptorchidism.

PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice.

It was reported that 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide and a possible endocrine disruptor, can disturb spermatogenesis, but the precise mechanism is not understood. Since 2,4-D is a weak peroxisome proliferator in hepatocytes and peroxisome proliferator-activated receptor α (PPARα) is also expressed in Leydig cells, this study aimed to investigate the link between PPARα and 2,4-D-mediated testicular dysfunction. 2,4-D (130 mg/kg/day) was administered to wild-type and Ppara-null mice for 2 weeks, and the alterations in testis and testosterone/cholesterol metabolism in Leydig cells were examined. Treatment with 2,4-D markedly decreased testicular testosterone in wild-type mice, leading to degeneration of spermatocytes and Sertoli cells. The 2,4-D decreased cholesterol levels in Leydig cells of wild-type mice through down-regulating the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 and reductase, involved in de novo cholesterogenesis. However, the mRNAs encoding the important proteins involved in testosterone synthesis were unchanged by 2,4-D except for CYP17A1, indicating that exhausted cholesterol levels in the cells is a main reason for reduced testicular testosterone. Additionally, pregnancy rate and the number of pups between 2,4-D-treated wild-type male mice and untreated female mice were significantly lower compared with those between untreated couples. These phenomena were not observed in 2,4-D-treated Ppara-null males. Collectively, these results suggest a critical role for PPARα in 2,4-D-induced testicular toxicity due to disruption of cholesterol/testosterone homeostasis in Leydig cells. This study yields novel insights into the possible mechanism of testicular dysfunction and male infertility caused by 2,4-D.

Germ cell transplantation into mouse testes procedure.

OBJECTIVE: To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes. DESIGN: Video presentation of an animal model for research in reproductive and regenerative medicine. SETTING: Research laboratory. ANIMAL(S): Male nude mice (NU-Foxn1(nu)). INTERVENTION(S): Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells. MAIN OUTCOME MEASURE(S): Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin. RESULT(S): In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3 months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation. CONCLUSION(S): Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future.

Estrogen-dependent and -independent estrogen receptor-alpha signaling separately regulate male fertility.

Estrogen receptor-alpha (ERalpha) plays a critical role in male reproductive tract development and fertility. To determine whether estrogen-dependent and -independent ERalpha mechanisms are involved in male fertility, we examined male estrogen nonresponsive ERalpha knock-in mice. These animals have a point mutation (G525L) in the ligand-binding domain of ERalpha that significantly reduces interaction with, and response to, endogenous estrogens but does not affect growth factor activation of ligand-independent ERalpha pathways. Surprisingly, we found that ligand-independent ERalpha signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption. In contrast, estrogen-dependent ERalpha signaling is required for germ cell viability, most likely through support of Sertoli cell function. By treating estrogen nonresponsive ERalpha knock-in (ENERKI) mice with the ERalpha selective synthetic agonist propyl pyrazole triol, which is able to bind and activate G525L ERalpha in vivo, we discovered male fertility required neonatal estrogen-mediated ERalpha signaling. Thus, our work indicates both estrogen-dependent and -independent pathways play separable roles in male murine reproductive tract development and that the role of ERalpha in human infertility should be examined more closely.

Proliferation and apoptosis of spermatogonia in postpuberal boar (Sus domesticus) testes with spontaneous unilateral and bilateral abdominal cryptorchidism.

Cryptorchidism is a frequent male sexual disorder in mammals, which affects the histology of the tunica propria, interstitial tissue, blood vessels, seminiferous epithelium and testis functioning. In this paper, proliferation and apoptosis were examined in the seminiferous epithelium of both testes from unaffected boars and from boars suffering unilateral and bilateral cryptorchidism. In germ cells, proliferation was studied using the immunohistochemical PCNA technique, and apoptosis was analysed by in situ TUNEL labelling. An index was obtained for the proliferation and apoptosis observed in seminiferous tubules. In abdominal testes the epithelium contained few spermatogonia and Sertoli cells. In the testes of unaffected boars, numerous spermatogonia proliferated, whereas in cryptorchid testes such proliferation was lower and the proliferation/apoptosis ratio diminished. In the unaffected group, the TUNEL-positive germ cells were spermatogonia and spermatocytes in different phases of meiosis. In abdominal testes, the TUNEL-positive germ cells were spermatogonia alone. The apoptosis index of both abdominal and scrotal testes was similar. In conclusion, spontaneous cryptorchid testes showed a lower rate of spermatogonia proliferation in the seminiferous epithelium.

Inhibin B: a marker for the functional state of the seminiferous epithelium in patients with azoospermia factor C microdeletions.

Testicular production of inhibin B is believed to be dependent on the presence of germ cells within the seminiferous tubules. However, this association has recently been questioned in patients with deletions of azoospermia factor (AZF) on the Y chromosome. We have addressed this problem in 442 unselected infertile/subfertile patients (excluding obstructive and iatrogenic forms) who were analyzed for Yq microdeletions. AZFc microdeletions were found in 16 patients (3.8% of the total infertile group, but 9% of the subgroup with azoospermia or severe oligozoospermia with sperm concentration <1 x 10(6)/ml). The reproductive hormone profiles in patients with AZFc microdeletions were analyzed and compared with those in infertile patients without microdeletions and those in fertile control individuals. The mean serum inhibin B concentration in the patients with AZFc microdeletions (39.5 +/- 36.0 pg/ml) was significantly lower than that in the group of infertile patients without microdeletions (134.6 +/- 88.5 pg/ml). However, no significant difference was found compared with that in a matched group of infertile patients with comparably low sperm counts (72.6 +/- 75.5 pg/ml). Bilateral testicular biopsies in the AZFc-deleted patients revealed a variable histological pattern suggestive of a progressive depletion of seminiferous epithelium. An association between testicular pathology and the reproductive hormone profile was found; the more severe forms had lower inhibin B and higher FSH levels. Importantly, if Sertoli cell-only tubules were prevalent in the biopsy, inhibin B was invariably undetectable. In patients with bilateral spermatocytic arrest, inhibin B remained within the normal range, which is consistent with a role of spermatocytes in the maintenance of inhibin B secretion. Our data support the view that, in contrast to recently published data, in patients with AZF microdeletions the serum concentration of inhibin B is dependent upon the functional interaction between Sertoli cells and spermatocytes and/or spermatids.

Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis.

This study investigates the role of neutrophils in ischemia-induced aspermatogenesis in the mouse. Previous studies in the rat have demonstrated that ischemia-inducing testicular torsion followed by torsion repair and reperfusion resulted in germ cell-specific apoptosis. This was correlated with an increase in neutrophil adhesion to subtunical venules, an increase in reactive oxygen species, and increased expression of several apoptosis-associated molecules. In the present investigation, wild-type C57BL/6 mice were subjected to various degrees and duration of testicular torsion. A torsion of 720 degrees for 2 h caused disruption of the seminiferous epithelium and significantly reduced testis weight and daily sperm production. An immunohistochemical method specific for apoptotic nuclei indicated that these effects were due to germ cell-specific apoptosis. An increase in myeloperoxidase (MPO) activity and an increase in the number of neutrophils adhering to testicular subtunical venules after torsion repair/reperfusion demonstrated an increase in neutrophil recruitment to the testis. In contrast, E-selectin knockout mice and wild-type mice rendered neutropenic showed a significant decrease in neutrophil recruitment as evidenced by MPO activity and microscopic examination of subtunical venules. Importantly, germ cell-specific apoptosis was also reduced. Thus, germ cell-specific apoptosis is observed after ischemia/reperfusion of the murine testis, and this apoptosis is directly linked to the recruitment of neutrophils to subtunical venules. Endothelial cell adhesion molecules, particularly E-selectin, play an important role in mediating this pathology.

Histología testicular humana comparada, adulto joven y senil / Human testicular histology in young and senile men

La fisiología del envejecimiento implica cambios morfológicos que interfieren con la función testicular normal. El hombre senil mantiene la potencia fecundante, aunque su eficacia disminuye. Posiblemente, asociada a una presentación histopatológica testicular. Se utilizaron muestras de tejidos testicular de 3 individuos seniles (69 años), sometidos a orquiectomía terapéutica y de un joven adulto (25 años). Fallecido por causa desconocida. Las gónadas fueron procesadas por técnicas histológicas para Hematoxina-P.A.S. Al microscopio se evaluaron la morfometría, celularidad e histopatología del epitelio seminífero. En los resultados se obtuvo una reducción del diámetro tubular y altura del epitelio seminífero en los individuos seniles, al ser comparados con el individuo joven. Paralelamente, en los testículos de individuos seniles se describio una drástica disminución en el número de células de De Sertoli, y un poco menor para espermatogonias tipo A oscuras, A claras y B; con una relación gonia/Sertori alterada. Asociado a lo anterior, se encontró un porcentaje reducido de espermátidas redondas y alargadas y de espermatozoides, en lumen por túbulo. La evaluación histopatológica en los individuos seniles reveló un epitelio seminífero severamente dañado, con presencia de vacuolización, discontinuidad del epitelio y detención de la espermatogénesis. Por lo tanto, se concluye la existencia de un notorio efecto adverso del progreso de la edad hacia senil, en la estructura histológica testicular, involucrando las diferentes poblaciones celulares y la relación entre ellas, como también la integridad del epitelio seminífero

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Does ethane 1,2-dimethanesulphonate (EDS) have a direct cytotoxic effect on the seminiferous epithelium of the rat testis?

The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.