Imatinib reduces the fertility of male mice by penetrating the blood-testis barrier and inducing spermatogonia apoptosis

Imatinib, the first generation of tyrosine kinase inhibitor, is used to treat and improve the prognosis of chronic myelogenous leukemia (CML). Clinical data suggest that imatinib could cross the blood-testis barrier and reduces the fertility of patients with CML-chronic phase. However, its exact molecular mechanism has not been fully elucidated. In this study, adult male Kunming mice were treated with different doses of imatinib for 8 weeks. The fertility was evaluated, and the sex hormone levels in the blood were detected by enzyme-linked immunosorbent assay. Histological changes were detected by hematoxylin and eosin staining. The concentration of imatinib in semen and blood was detected by liquid chromatography-mass spectrometry. The ultrastructure of blood-testis barrier and apoptotic bodies were observed by transmission electron microscope. The expression of blood-testis barrier function-regulating protein, Mfsd2a, and apoptosis-associated proteins in testis tissue was detected by immunohistochemistry and Western blot. The results indicated that the fertility of male mice was significantly decreased in a dose-dependent manner after imatinib treatment. Certain hormones in the serum were increased in imatinib treatment groups. Sperm morphology and testicular tissue showed various changes after imatinib treatment. The blood-testis barrier was destroyed and the concentration of imatinib in semen was similar to that in blood after imatinib treatment. Apoptosis was significantly increased in testis tissue after imatinib treatment. Collectively, these results suggest that imatinib can alter blood-testis barrier function, induce apoptosis of spermatogonia, and adversely affect fertility by reducing the number of spermatozoa, decreasing sperm motility and increasing the deformity rate.     

Kindlin-2 in Sertoli cells is essential for testis development and male fertility in mice

Kindlin-2 is known to play important roles in the development of mesoderm-derived tissues including myocardium, smooth muscle, cartilage and blood vessels. However, nothing is known for the role of Kindlin-2 in mesoderm-derived reproductive organs. Here, we report that loss of Kindlin-2 in Sertoli cells caused severe testis hypoplasia, abnormal germ cell development and complete infertility in male mice. Functionally, loss of Kindlin-2 inhibits proliferation, increases apoptosis, impairs phagocytosis in Sertoli cells and destroyed the integration of blood-testis barrier structure in testes. Mechanistically, Kindlin-2 interacts with LATS1 and YAP, the key components of Hippo pathway. Kindlin-2 impedes LATS1 interaction with YAP, and depletion of Kindlin-2 enhances LATS1 interaction with YAP, increases YAP phosphorylation and decreases its nuclear translocation. For clinical relevance, lower Kindlin-2 expression and decreased nucleus localization of YAP was found in SCOS patients. Collectively, we demonstrated that Kindlin-2 in Sertoli cells is essential for sperm development and male reproduction.Subject terms: Apoptosis, Male factor infertility     

Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis

Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.     

Diet-Induced Obesity in Male C57BL/6 Mice Decreases Fertility as a Consequence of Disrupted Blood-Testis Barrier

Obesity is a complex metabolic disease that is a serious detriment to both children and adult health, which induces a variety of diseases, such as cardiovascular disease, type II diabetes, hypertension and cancer. Although adverse effects of obesity on female reproduction or oocyte development have been well recognized, its harmfulness to male fertility is still unclear because of reported conflicting results. The aim of this study was to determine whether diet-induced obesity impairs male fertility and furthermore to uncover its underlying mechanisms. Thus, male C57BL/6 mice fed a high-fat diet (HFD) for 10 weeks served as a model of diet-induced obesity. The results clearly show that the percentage of sperm motility and progressive motility significantly decreased, whereas the proportion of teratozoospermia dramatically increased in HFD mice compared to those in normal diet fed controls. Besides, the sperm acrosome reaction fell accompanied by a decline in testosterone level and an increase in estradiol level in the HFD group. This alteration of sperm function parameters strongly indicated that the fertility of HFD mice was indeed impaired, which was also validated by a low pregnancy rate in their mated normal female. Moreover, testicular morphological analyses revealed that seminiferous epithelia were severely atrophic, and cell adhesions between spermatogenic cells and Sertoli cells were loosely arranged in HFD mice. Meanwhile, the integrity of the blood-testis barrier was severely interrupted consistent with declines in the tight junction related proteins, occludin, ZO-1 and androgen receptor, but instead endocytic vesicle-associated protein, clathrin rose. Taken together, obesity can impair male fertility through declines in the sperm function parameters, sex hormone level, whereas during spermatogenesis damage to the blood-testis barrier (BTB) integrity may be one of the crucial underlying factors accounting for this change.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation

Meiotic recombination is initiated by a series of double-strand breaks (DSBs) in areas of the genome that generally contain promoters and feature an open chromatin configuration [T.D. Petes, Meiotic recombination hot spots and cold spots, Nat. Rev. Genet. 2 (2001) 360-369]. To investigate whether induced DSBs likewise lead to recombinational repair and whether the placement of new exchange events alters normal patterns of recombination, we used the chemotherapeutic drug cisplatin (CP) to generate additional DSBs throughout the mouse genome. Treatment with CP impaired spermatogenesis, as exhibited by reductions in sperm counts, reductions in both testicular size and weight, changes in the distribution of cells at various prophase I substages, prolonged increases in germ cell apoptosis, and an increased incidence of synaptic abnormalities. Unexpectedly, however, no obvious effect on genome-wide recombination levels in CP-treated animals was observed, nor was the level of aneuploidy increased in sperm from exposed males.     

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.     

Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells

Vitamin C is an essential micronutrient for the development of male germ cells. In the gonad, the germ cells are isolated from the systemic circulation by the blood-testis barrier, which consists of a basal layer of Sertoli cells that communicate through an extensive array of tight junction complexes. To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in this barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells. To date, there is no available information on the mechanism of vitamin C transport across the blood-testis barrier. This work describe the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules. RT-PCR analyses revealed that 42GPA9 cells express both vitamin C transport systems, a finding that was confirmed by immunocytochemical and immunoblotting analysis. The kinetic assays using radioactive vitamin C revealed that both ascorbic acid (AA) transporters, SVCT1 and SVCT2, are functionally active. Moreover, the kinetic characteristics of dehydroascorbic acid (DHA) and 3-methylglucose (OMG) transport by 42GPA9 Sertoli cells correspond to facilitative hexose transporters GLUT1, GLUT2 and GLUT3 expressed in these cells. This data is consistent with the concept that Sertoli cells have the ability to take up vitamin C. It is an important finding and contributes to our knowledge of the physiology of male germ cells.     

Melatonin ameliorates paclitaxel‐induced mice spermatogenesis and fertility defects

Chemotherapeutic drug of paclitaxel (PTX) has been shown to cause reproductive toxicity thus affecting male fertility, but its underlying molecular basis is unclear. Melatonin (MLT) can mitigate the reproductive damage caused by certain chemotherapy drugs. In this study, we aimed to identify impact of PTX on the main biological processes and protective effect of MLT on reproductive damage caused by PTX. Mice exposed to PTX mainly impaired spermatogenesis, such as decreased sperm counts, reduced sperm motility and increased abnormal sperm. Decreased expressions of germ cell proliferation‐associated protein PCNA and meiosis‐related protein SYCP3 induced by PTX were determined by Western blot, while MLT ameliorated this effect and increased the expressions of PCNA, SYCP3, DMC1, STRA8 and fertility‐related protein of HSPA2, resulting in significantly improved spermatogenesis and sperm quality levels. In vitro fertilization experiment showed that PTX significantly decreased blastocyst formation rates, which can be improved by MLT administration, but not two‐cell development rates. Taken together, this work demonstrated PTX can adversely affect germ cell proliferation and meiosis, which ultimately influence sperm quality and male fertility, and highlighted the protective ability of MLT on ameliorating the side effects of PTX, especially on sperm quality. The results provide information to further the study on the molecular mechanism of PTX''s effects on male reproduction and the protective mechanism of MLT.     

Cyclic formation and decay of the blood-testis barrier in the mink (Mustela vison), a seasonal breeder

The correlations between the germ cell population and the blood-testis barrier were studied during puberty and throughout the reproductive cycle in a seasonal breeder, the mink. A classification of 12 stages, corresponding to the cellular associations appearing during the cycle of the seminiferous epithelium, was proposed and used to identify the stages of the cycle in pubertal mink. In adult mink, the reproductive cycle was divided into two spermatogenic phases--an active phase lasting 9 months, and an inactive phase lasting 3 months. The active spermatogenic phase was broken down into three distinct periods: the first spermatogenic wave, the peak of spermatogenic activity, and the last spermatogenic wave. Degenerating germ cells were found in comparable and relatively low proportions during puberty and during the first and last spermatogenic waves of the adult reproductive cycle. The permeability of the blood-testis barrier to intravascularly infused electron-opaque tracers (i.e., horseradish peroxidase and lanthanum) was tested at the time of the first spermatogenic wave at puberty and throughout the reproductive cycle of the adult. The relationship between epithelial permeability and germ cell populations prevailing during puberty and during the first and last spermatogenic waves of the adult active phase was the same. During puberty, the establishment of the blood-testis barrier did not coincide with the appearance of a particular step of meiosis but was correlated with the development of a tubular lumen. In adult mink, the barrier cyclically decayed during the last wave of the active spermatogenic phase and reformed during the first wave of the next active phase. The decay and the reformation of the barrier were not coincident with the appearance or disappearance of a particular generation of the germ cell population from the seminiferous epithelium but were correlated with cyclic cytological changes in Sertoli cells and the rhythmic development and occlusion of the lumen. During the peak months of the active spermatogenic phase, however, a blood-testis barrier secluded spermatogonia and young spermatocytes from older generations of germ cells. It is concluded that during puberty and also during the first and last spermatogenic wave of the adult mink reproductive cycle, the development of germ cells is possible in the absence of a competent, impermeable blood-testis barrier, and the transient presence of a permeable epithelial barrier does not initiate an autoimmune response of sufficient magnitude to cause destruction of the seminiferous epithelium.     

Size-selective junctional barrier and Ca(2+)-independent cell adhesion in the testis of Cynops pyrrhogaster: expression and function of occludin

In urodeles which has testicular structure different from that in mammals, blood-testis barrier was reported to exist like in mammals. However, molecular and functional analyses of the components of the blood-testis barrier in urodeles have not been reported yet. Toward elucidation of the barrier functions and their molecular components in newt testis, we aimed to isolate occludin cDNAs and obtained two kinds of occludin partial cDNAs (occludin 1 and 2) encoding the putative second extracellular loop. Immunoblot and immunofluorescence studies using antibodies against peptides each corresponding to a part of the second extracellular loop of occludin 1 and 2, and those against beta-catenin and zonula occludens-1 (ZO-1) showed that occludin, as well as beta-catenin and ZO-1, was expressed not only in Sertoli cells but also in germ cells throughout all the stages from spermatogonia to elongate spermatids. Tracer experiments revealed a size-selective barrier which allows small molecules ( approximately 500 Da) to get into cysts through Sertoli cells' barrier, but not larger ones (>1.9 kDa) in the stages from spermatogonia to almost mature sperm. No occludin peptides corresponding to a part of the second extracellular loop destroyed the junctional barrier, while both the peptides and antibodies significantly inhibited reaggregation of the dissociated testicular cells which was to a large extent Ca(2+)-independent. These results indicate that the second extracellular loop of occludin is involved in cell adhesion rather than in size-selective barrier in newt testis, though the possibility cannot be excluded that the peptides were not long enough to inhibit the barrier function.     

Cellular suppression of murine ADCC and NK activities induced by Corynebacterium parvum

Summary Administration of a single dose of C. parvum (CP) induces depression of splenic NK activity in mice after a lag period of 3–5 days and this depression lasts about 2 weeks. The depressed levels of NK activity noted in this study depended on time of CP administration and were associated with the induction of suppressor cell activity. Neonatally thymectomized or sublethally irradiated mice had unimpaired ability to generate suppressor cells following CP treatment. Depletion of adherent/phagocytic cells by carbonyl iron plus magnetism, Sephadex G-10 filtration, or both neither enriched NK activity nor removed suppressor activity from the spleens of CP-treated mice. Antibody-dependent cellular cytotoxicity (ADCC) against lymphoma targets was also depressed in CP-treated mice, accompanied by a concomitant appearance of suppressor cells that interfere with ADCC at the effector level.     

CENP-B is not critical for meiotic chromosome segregation in male mice.

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P> or = 0.5) or from 129/Swiss isogenic mice (P> or = 0.5) and B6C3F1 mice (P> or = 0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.     

NANOS2 promotes male germ cell development independent of meiosis suppression

NANOS2 is an RNA-binding protein essential for fetal male germ cell development. While we have shown that the function of NANOS2 is vital for suppressing meiosis in embryonic XY germ cells, it is still unknown whether NANOS2 plays other roles in the sexual differentiation of male germ cells. In this study, we addressed the issue by generating Nanos2/Stra8 double knockout (dKO) mice, whereby meiosis was prohibited in the double-mutant male germ cells. We found that the expression of male-specific genes, which was decreased in the Nanos2 mutant, was hardly recovered in the dKO embryo, suggesting that NANOS2 plays a role in male gene expression other than suppression of meiosis. To investigate the molecular events that may be controlled by NANOS2, we conducted a series of microarray analyses to search putative targets of NANOS2 that fulfilled 2 criteria: (1) increased expression in the Nanos2 mutant and (2) the mRNA associated with NANOS2. Interestingly, the genes predominantly expressed in undifferentiated primordial germ cells (PGCs) were significantly selected, implying the involvement of NANOS2 in the termination of the characteristics of PGCs. Furthermore, we showed that NANOS2 is required for the maintenance of mitotic quiescence, but not for the initiation of the quiescence in fetal male germ cells. These results suggest that NANOS2 is not merely a suppressor of meiosis, but instead plays pivotal roles in the sexual differentiation of male germ cells.