Pentacle gold–copper alloy nanocrystals: a new system for entering male germ cells in vitro and in vivo

ABSTRACT Gold-based nanocrystals have attracted considerable attention for drug delivery and biological applications due to their distinct shapes. However, overcoming biological barriers is

a hard and inevitable problem, which restricts medical applications of nanomaterials _in vivo_. Seeking for an efficient transportation to penetrate biological barriers is a common need.

There are three barriers: blood-testis barrier, blood-placenta barrier, and blood-brain barrier. Here, we pay close attention to the blood-testis barrier. We found that the pentacle

gold–copper alloy nanocrystals not only could enter GC-2 cells _in vitro_ in a short time, but also could overcome the blood–testis barrier and enter male germ cells _in vivo_. Furthermore,

we demonstrated that the entrance efficiency would become much higher in the development stages. The results also suggested that the pentacle gold–copper alloy nanocrystals could easier

enter to germ cells in the pathological condition. This system could be a new method for theranostics in the reproductive system. SIMILAR CONTENT BEING VIEWED BY OTHERS DEVELOPMENT OF A

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February 2024 INTRODUCTION Gold-based nanocrystals have been widely used in drug delivery and other biological applications due to their advantageous biocompatibility, plasmonic

properties1,2,3,4,5,6. However, biological barriers are the stumbling blocks to nanomaterials in biomedical applications7,8,9. Therefore, searching for a successful delivery system to

achieve fast and efficient transportation to penetrate biological barriers is the only way which must be passed. Our previous work reported the synthesis of pentacle gold–copper nanocrystals

as a new class of gold-based nanostructures with their low cost of alloying and high photothermal performance10. Using a remarkably simple co-reduction synthetic route, gold and copper

precursors can be made to generate a starfish-like nanostructure with five long branches, which are used for tumor photothermal therapy. Because of their sharp arms, the pentacle

nanocrystals may find distinctive use in applications related to penetrating some biological barriers. Blood–testis barrier, blood–placenta barrier, and blood–brain barrier are three

biological barriers which prevent nanomaterial applications in medical fields11. Here, we investigated the blood–testis barrier. Spermatogenesis in the testes is a coordinated process, which

includes spermatogonial stem cell division into spermatogonia, and diploid spermatogonia differentiate into meiotic spermatocytes, which divide twice without additional DNA replication,

producing haploid round spermatids12,13,14,15,16. Only two cell types constitute the seminiferous epithelium, the Sertoli and the germ cells. Spermatogenesis occurs in a very safe

environment because of the blood–testis barrier (BTB), which is formed by adjacent Sertoli cells at the basal compartment of the seminiferous epithelium17,18,19,20,21,22. In theory,

nanoparticles cannot penetrate the BTB, which prevents nanoparticles producing toxicity in male germ cells17. However, several studies have shown that nanoparticles can penetrate the BTB

while non-nanoparticles can not17. In recent years, some reports have indicated biodistribution of gold nanoparticles in the testes in both time-dependent and size-dependent manners23,24.

The previous reports indicate that route of administration, dose of AuNP and AuNP characteristics (e.g., size, shape, chemical composition and surface charge) play a significant role in

determining the reproductive nanotoxicity of AuNP on spermatogenesis25,26. The shape of AuNP is an important characteristic, which may determinate the biodistribution or biotoxicity on

spermatogenesis. Our previous work reported pentacle gold-copper nanocrystals were a potent agent for tumor therapy10. Therefore, it is important and necessary to study the biocompatibility,

biological effects and biomedical application of the pentacle gold-copper alloy nanocrystals deeply. However, to our knowledge, no other reports have been published about testicular

biodistribution or bioapplication of pentacle gold–copper alloy nanocrystals. Herein, we have found that pentacle gold–copper alloy nanocrystals entered GC-2 cells (a spermatocyte cell line)

within a short time _in vitro_, and showed the ability of pentacle nanocrystals to penetrate the BTB _in vivo_. We have also investigated a way to increase the amount of pentacles in the

testes. In addition, the pentacles were combined with fluorescein isothiocyanate isomer I (FITC) as a nanocomplex model to assess their potential for carrying siRNA/miRNA/DNA or polypeptides

into male germ cells _in vivo_ to solve issues of male infertility. RESULTS PENTACLE GOLD–COPPER ALLOY NANOCRYSTALS ENTER GC-2 CELLS _IN VITRO_ WITHOUT CYTOTOXICITY Pentacle gold–copper

alloy nanocrystals were synthesized through a modified strategy in our previous study in methods10. Figure 1a shows a transmission electron microscope (TEM) image of pentacles, indicating

that most of the nanocrystals are five-fold twined structures with sharp arms. To stabilize the structure and prolong blood circulation, we decorated the pentacles with thiolated

poly(ethylene glycol) (PEG) for future use. Many studies have demonstrated that the uptake of gold nanoparticles by mammalian cells is size- and shape-dependent27,28,29,30. To investigate

the uptake of pentacles in mammalian reproductive cells, the as-synthesized pentacle-PEG was added to GC-2 cells. After incubating for different times, the accumulation of pentacle-PEG in

GC-2 cells was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Fig. 1b). The results show that PEG-modified pentacle gold–copper alloy nanocrystals entered GC-2 cells

after a short time. The maximum uptake appeared at 6 h, and then the amount of pentacles decreased about 30% at 12 h. Also, we found pentacles after 3 h in TEM images, which further

demonstrated that pentacle-PEG entered GC-2 cells within a short time (Fig. 1c–f). The cytotoxicity of pentacle gold–copper alloy nanocrystals of different concentrations to the GC-2 cell

line was investigated. We found there were no significant cytotoxicity at 15, 30, 60 μg ml−1 pentacle NCs after 24 h (Supplementary data, Figure S1). The pentacle-PEG did not appear to be

toxic at 15 μg mL−1 (gold atom concentration) measured by a standard MTT assay (Fig. 2a) at 12, 24 and 36 h. Also, we double stained GC-2 cells with calcein-AM for living cells and with the

nuclear stain propidium iodide (PI) as an indicator of dead cells. There was no significant difference in cell death between pentacle-PEG treatment and the phosphate buffer solution (PBS)

group (Fig. 2b). These results suggest that the pentacles do not have an impact on cell proliferation and death, indicating good biocompatibility with mammal reproductive cells _in vitro_.

To simulate a nanocomplex system, we labeled pentacles with FITC. We exposed FITC-labeled pentacles to GC-2 cells for 0, 3, 6 and 12 h. In contrast, the same concentration of FITC was also

incubated with GC-2 cells. Figure 3 shows the results of fluorescence emission detection. There was no green fluorescence except for the pentacle-FITC group, which demonstrated that the

pentacles could deliver FITC molecules to GC-2 cells efficiently. In the pentacle-FITC group, the quasi-round structures with green fluorescence were assigned as endocytic vesicles which

took pentacles to the cytomembrane of GC-2 cells. Consistent with the ICP-MS results in Fig. 1b, the highest fluorescent signal in the pentacle-FITC group appeared at 6 h treatment, further

demonstrating that the pentacles entering GC-2 cells was a fast procedure. The results suggested the pentacles could deliver biological molecules efficiently to GC-2 cells _in vitro_.

PENTACLE GOLD–COPPER ALLOY NANOCRYSTALS ENTER MALE GERM CELLS OF ADULT MOUSE _IN VIVO_ Although pentacles can be taken up by GC-2 cells _in vitro_, it is also possible for the BTB to prevent

pentacles entering male germ cells _in vivo_. It is an important issue to study whether the pentacle gold–copper alloy nanocrystals can penetrate the BTB as expected. In the present study,

we firstly used FITC-labeled pentacles to investigate whether they can penetrate the BTB with the conjunct organic molecules. We directly injected PBS, free FITC or FITC-labeled pentacles

into mouse testes _in situ_, which meant it was injected into the interspace of the seminiferous tubules outside the BTB. We collected testes of adult male mice and stretched the

seminiferous tubules on the slide at each time point. As shown in Fig. 4, the fluorescence images in the FITC group are similar to that of the PBS group, indicating that almost all the free

FITC molecules are blocked by the BTB. In contrast, the green fluorescent signal of the pentacle-FITC group was high enough to fill the overall structure of the seminiferous tubules in the

chosen images. The results demonstrated that the pentacles could pass through the BTB. We also carried out the experiment by intravenous (i.v.) injection to male mice. PBS and pentacle-PEG

were injected via the tail vein once, respectively. 3 and 24 h after injection, the accumulation of pentacles in testes was analyzed by ICP-MS. The results show that approximately 2.5 and

6.6 ng of pentacle nanocrystals were found in the testes 3 and 24 h after i.v. injection, respectively (Fig. 5). Although the amount of pentacles in the testes was small, the increasing

trend suggests that, with repeated exposure, more pentacle nanocrystals would be expected to accumulate in the testes. They could potentially pass through the BTB. Also, the male

reproductive toxicity was investigated; as shown in Fig. 6, there were no significant differences in testicular morphology between pentacle-treated mice and the control group. All the

results suggest that the pentacles could penetrate the BTB and have good biocompatibility _in vivo_. We have proven that the pentacle nanocrystals can pass through the BTB, but the BTB

prevents more pentacles entering the seminiferous epithelium and entering the male germ cells. The results still show that the amount of pentacle nanocrystals in the testes was small, which

was consistent with other reported results26,31. Therefore, methods and conditions for increasing the amount of pentacles passing the BTB should be investigated. IN PUBERTY OR PATHOLOGICAL

CONDITIONS, INCREASING THE AMOUNT OF PENTACLES GOLD–COPPER ALLOY NANOCRYSTALS IN THE TESTES During development stages, the BTB is gradually formed by Sertoli cells. The BTB has been reported

to be established in rats by 15–18 days postpartum (ppd)32,33. In puberty, harmful substances in the environment can more easily enter germ cells. In the present study, 8 and 18 ppd rats

were intraperitoneally (i.p.) injected with PBS or PEG-modified pentacle gold–copper alloy nanocrystals in a single dose. The accumulation of pentacle nanocrystals in the testes was analyzed

by ICP-MS. As shown in Fig. 7, approximately 1.35 ng mg−1 (Au–Cu/testis weight) and 2.97 ng mg−1 of pentacles were found in the testes 24 h after i.p. injection, respectively. It is about

50 ng/g (Au–Cu/testis weight) in adult mice testes (Fig. 5), which was above 2000 ng/g (Au–Cu/testis weight) in puberty rat testes (Fig. 7). These results indicate that the pentacle

nanocrystals can enter the male germ cells in large amounts in juvenile rats. Also, we found branched nanocrystals in spermatocytes of 8 ppd rats from the TEM image (Supplementary data,

Figure S2), resembling the arms of a whole pentacle. In puberty, the pentacles can enter germ cells in large amounts in normal physiological conditions, which suggests that we can use the

pentacles as a delivery system for intervening in and treating the diseases of male germ cells _in vivo_. The BTB serves as a “gate-keeper” that regulates proteins and/or biomolecules that

can enter the adluminal compartment from the interstitium34,35. However, the BTB is not an impervious barrier. During normal physiological conditions of murine spermatogenesis,

preleptotene/leptotene spermatocytes transit and migrate from the basal to the adluminal compartment through the BTB during stages VIII–IX, in which the BTB has transitory

permeability36,37,38,39. In some abnormal conditions, many materials and treatments such as adjudin, cadmium, and hot baths can also destroy the BTB and enhance its

permeability40,41,42,43,44,45,46. For example, when treated in a hot bath, the BTB will be destroyed and exogenous materials will enter germ cells more easily. In the present study, we

treated the mice at 43 °C for 10 min to mimic the pathological condition cryptorchidism, which destroyed the BTB; 24 h after hot bath treatment, we directly injected PBS, free FITC or

FITC-labeled pentacle gold–copper alloy nanocrystals into mouse testes _in situ_. The fluorescence images show that the fluorescent signal of FITC-labeled pentacle gold–copper alloy

nanocrystals within hot bath treatment was much higher than in groups at room temperature treatment (Supplementary data, Figure S3). The results suggest that the pentacle gold–copper alloy

nanocrystals can more easily enter germ cells in pathological conditions. Therefore, if the BTB has been formed stably, we can use pentacles as delivery vehicles and cure the diseases of

male germ cells under heat treatment _in vivo_. DISCUSSION Through shape control, the synergy of combining gold and copper metals can be better exploited. We have reported an aqueous phase

route to the synthesis of pentacle gold–copper alloy nanocrystals with five fold twinning, the size of which can be tuned in the range from 45 to 200 nm. The pentacle gold–copper alloy

nanocrystals showed plasmonic and catalytic properties, and had notable photothermal effect to tumours in our previous study. In the present, we investigated whether the pentacle gold–copper

alloy nanocrystals cause harmful impact on the male reproductive system. We exploited pentacle gold–copper alloy nanocrystals to enter GC-2 cells _in vitro_ and penetrate the BTB _in vivo_.

We demonstrated that the PEG-modified pentacle gold–copper alloy nanocrystals can enter GC-2 cells within a short time, and have good biocompatibility with GC-2 cells _in vitro_. Also, we

proved that the pentacle gold–copper alloy nanocrystals can pass through the BTB in adult male mice, and found ways to improve the efficiency of entrance to the testes. In order to simulate

delivery circumstances, FITC-labeled pentacle nanocrystals were also involved in the research. We further found that the pentacle nanocrystals can enter male germ cells in large amounts in

juvenile rats, suggesting that we can deliver drugs or biomolecules by the nanoparticles at this stage. For example, some genetic factors can cause infertility. We can deliver the genes and

siRNA or CRISPR/Cas9 (a gene editing system) into the testis through destroying or re-establishing the BTB at the puberty. Our results could provide a method for high-branched nanocrystals

to be applied to theranostics in the genital system. METHODS SYNTHESIS OF PENTACLE GOLD–COPPER ALLOY NANOCRYSTALS In a standard synthesis of pentacle gold–copper alloy nanocrystals, 0.33 mL

of aqueous CuCl2·2H2O (100 mM), 0.27 mL of aqueous HAuCl4·3H2O (100 mM), 45 mg of HDA, 1.4 mL of aqueous glucose (1 M) and 3 mL of water were added to a 20 mL vial at room temperature. After

the vial had been capped, the solution was magnetically stirred at room temperature overnight. The capped vial was then transferred into an oil bath and heated at 100 °C for 15 min under

magnetic stirring. As the reaction proceeded, the solution changed its color from Kelly green to brown. To prepare samples for electron microscopy characterization, the pentacle nanocrystals

were centrifuged at 10,000 rpm for 8 min and washed by water three times and ethanol twice to remove excess precursor, HDA and glucose. PEG AND FITC MODIFIED PENTACLES In a typical process,

10 mg of HS-PEG-NH2 was added to an aqueous suspension of Au-Cu pentacles (1 mg/mL, 5 mL). The reaction mixture was vortexed immediately and then magnetic stirred overnight, followed by

centrifuging at 14000 rpm for 5 min to remove excess PEG. For FITC-pentacles, 0.1 mL of 1 mM FITC solution was added to 5 mL of HS-PEG-NH2 functionalized pentacle solution. The mixture was

magnetic stirred for several hours. Then the final solution was centrifuged at 10,000 rpm for 8 min and washed by water three times to remove dissociative FITC. CELL CULTURE GC-2 cells were

grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies Inc.) and 1% antibiotics (100 Units mL−1 penicillin and 100 mg mL−1 streptomycin; Life

Technologies Inc.) and cultured at 37 °C with 5% CO2. ANIMALS Animals were handled according to the guidelines of experimental animal ethics committee of Anhui medical university, and the

study was reviewed and approved by the experimental animal ethics committee of Anhui medical university. ICP-MS _In v_itro, the 6-well plate GC-2 cells were exposed to PEG-modified 100 nm

pentacle gold–copper alloy nanocrystals at 15 μg mL−1 for 0, 3, 6 and 12 h. At each time point, the cells were counted, collected and dissolved in aqua regia. The amount of gold in the

testes was measured by ICP-MS (Atomscan Advantage, Thermo Jarrell Ash Corporation, USA). We used a simple model to calculate the pentacle nanocrystals by measured the ICP-MS results10. A

pentacle contains a decahedron and ten tetrahedra. Considering the average edge length of pentacle nanocrystals in the TEM image, the volume of a pentacle (_V_) can be calculated to be 26200

 nm3. According to our previous work, the pentacle nanocrystals were indexed as typical face-centered cubic (_fcc_) structure. Hence a cell contains 4 atoms. The volume of a unit cell (_V_0)

was calculated to be 0.064 nm3. Therefore, each pentacle contains _n_0 = 4 × _V_/_V_0 = 1.64 × 106 atoms. The average relative atomic mass (_M_) of the Au-Cu alloy pentacles was 178 (Au

86%, Cu 14%). Meanwhile, the total mass of the Au and Cu metals (_m_) was determined by ICP-MS. The total number of atoms (_n_) was _m_/_M_ * _N__A_, where NA represents Avogadro constant.

_In vivo_, for ICP-MS analyses, 6–8 week-old mice were i.v. injected with PBS or 12.5 mg kg−1 (by body weight) PEG-modified 100 nm pentacle gold–copper alloy nanocrystals once (single dose);

3 and 24 h after injection, the testes were collected, weighed and then dissolved in aqua regia. The amount of gold in the testes was measured by ICP-MS (Atomscan Advantage, Thermo Jarrell

Ash Corporation, USA). _In vivo_, for ICP-MS analyses, 8 and 18 ppd rats were i.p. injected with PBS or 25 mg kg−1 (by body weight) PEG-modified 100 nm pentacle gold–copper alloy

nanocrystals once (single dose); 24 h after injection, the testes were collected, weighed and then dissolved in aqua regia. The amount of gold in the testes was measured by ICP-MS. TEM _In

vitro_, the GC-2 cells were exposed to PEG-modified 100 nm pentacle gold–copper alloy nanocrystals at 15 μg mL−1 for 0, 3, 6 and 12 h. At each time point, the cells were collected, then

centrifuged, fixed with 2.5% glutaraldehyde solution for 6 h, dehydrated through an ethanol series (70% for 15 min, 90% for 15 min, and 100% for 15 min twice) and embedded in Epon/Araldite

resin (polymerization at 65 °C for 15 h). Thin sections were cut (70 nm thick), placed on grids and stained for 1 min each with 4% uranyl acetate (1:1, acetone/water) and 0.2% Reynolds lead

citrate (water), air-dried and imaged under an 80 kV JEOL-1230 TEM. _In vivo_, 8 and 18 ppd SD rats were i.p. injected with PBS or 25 mg kg−1 (by body weight) PEG-modified 100 nm pentacle

gold–copper alloy nanocrystals once (single dose); 24 h after injection, the testes were collected and fixed with 2.5% glutaraldehyde solution for 6 h. Finally, they were imaged under an 80 

kV JEOL-1230 TEM. FLUORESCENCE MICROSCOPY _In vitro_, the GC-2 cells were exposed to FITC-labeled 100 nm pentacle gold–copper alloy nanocrystals at 15 μg mL−1 for 0, 3, 6 and 12 h. In the

negative group, we treated GC-2 cells with PBS, and in the control group, we treated GC-2 cells with free FITC. The cells were washed twice with ice-cold PBS and fixed with 4%

paraformaldehyde for 20 min. Nuclei were stained with Hoechst 33342 (Sigma). Fluorescent signals were examined using a Nikon Eclipse 80i epifluorescence microscope. _In vivo_, PBS, free FITC

or 12.5 mg kg−1 (by body weight) FITC-labeled 100 nm pentacle gold–copper alloy nanocrystals were injected directly into mouse testes; 6 and 12 h after injection, the testes were cut, put

on the slides and then the seminiferous tubules were pulled by tweezers. The seminiferous tubules were stained with Hoechst 33342 (Sigma) and covered by a cover glass. Fluorescent signals

were examined using a Nikon Eclipse 80i epifluorescence microscope. STATISTICAL ANALYSIS Student’s t-test was applied to examine the differences among variables. Data are shown as mean ± SD,

unless otherwise indicated. _p_ values ≤0.05 were considered to be statistically significant. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Lin, Y. _et al_. Pentacle gold–copper alloy

nanocrystals: a new system for entering male germ cells _in vitro_ and _in vivo. Sci. Rep._ 6, 39592; doi: 10.1038/srep39592 (2016). PUBLISHER'S NOTE: Springer Nature remains neutral

with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES * Xia, Y. et al. Gold nanocages: from synthesis to theranostic applications. Acc Chem Res

44, 914–924 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Bardhan, R., Lal, S., Joshi, A. & Halas, N. J. Theranostic nanoshells: from probe design to imaging and treatment of

cancer. Acc Chem Res 44, 936–946 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Vijayaraghavan, P., Liu, C. H., Vankayala, R., Chiang, C. S. & Hwang, K. C. Designing

multi-branched gold nanoechinus for NIR light activated dual modal photodynamic and photothermal therapy in the second biological window. Adv Mater 26, 6689–6695 (2014). CAS  PubMed  Google

Scholar  * Cheng, K. et al. Construction and validation of nano gold tripods for molecular imaging of living subjects. J Am Chem Soc 136, 3560–3571 (2014). CAS  PubMed  PubMed Central 

Google Scholar  * Huang, P. et al. Triphase Interface Synthesis of Plasmonic Gold Bellflowers as Near-Infrared Light Mediated Acoustic and Thermal Theranostics. Journal of the American

Chemical Society 136, 8307–8313 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Song, J. et al. Ultrasmall Gold Nanorod Vesicles with Enhanced Tumor Accumulation and Fast Excretion

from the Body for Cancer Therapy. Adv Mater 27, 4910–4917 (2015). CAS  PubMed  Google Scholar  * Avila-Olias, M., Pegoraro, C., Battaglia, G. & Canton, I. Inspired by nature:

fundamentals in nanotechnology design to overcome biological barriers. Ther Deliv 4, 27–43 (2013). CAS  PubMed  Google Scholar  * Cao, C. et al. Targeted _in vivo_ imaging of microscopic

tumors with ferritin-based nanoprobes across biological barriers. Adv Mater 26, 2566–2571 (2014). CAS  PubMed  Google Scholar  * Blanco, E., Shen, H. & Ferrari, M. Principles of

nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol 33, 941–951 (2015). CAS  PubMed  PubMed Central  Google Scholar  * He, R. et al. Facile synthesis of

pentacle gold-copper alloy nanocrystals and their plasmonic and catalytic properties. Nat Commun 5, 4327 (2014). ADS  CAS  PubMed  Google Scholar  * Paul, Debbage & Gudrun, C. Thurner

Nanomedicine Faces Barriers. Pharmaceuticals 3 (2010). * Eddy, E. M. Male germ cell gene expression. Recent Prog Horm Res 57, 103–128 (2002). CAS  PubMed  Google Scholar  * Hecht, N. B.

Molecular mechanisms of male germ cell differentiation. Bioessays 20, 555–561 (1998). CAS  PubMed  Google Scholar  * Iguchi, N., Tobias, J. W. & Hecht, N. B. Expression profiling reveals

meiotic male germ cell mRNAs that are translationally up- and down-regulated. Proc Natl Acad Sci USA 103, 7712–7717 (2006). ADS  CAS  PubMed  PubMed Central  Google Scholar  * Liang, M. et

al. Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis. Sci Rep 4, 5966 (2014). CAS  PubMed  PubMed Central  Google Scholar  *

Cheon, Y. P. & Kim, C. H. Impact of glycosylation on the unimpaired functions of the sperm. Clin Exp Reprod Med 42, 77–85 (2015). PubMed  PubMed Central  Google Scholar  * Lan, Z. &

Yang, W. X. Nanoparticles and spermatogenesis: how do nanoparticles affect spermatogenesis and penetrate the blood-testis barrier. Nanomedicine-Uk 7, 579–596 (2012). CAS  Google Scholar  *

Dym, M. & Fawcett, D. W. The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 3, 308–326 (1970). CAS  PubMed  Google

Scholar  * Lui, W. Y., Mruk, D., Lee, W. M. & Cheng, C. Y. Sertoli cell tight junction dynamics: their regulation during spermatogenesis. Biol Reprod 68, 1087–1097 (2003). CAS  PubMed 

Google Scholar  * Al-Asmakh, M. & Hedin, L. Microbiota and the control of blood-tissue barriers. Tissue Barriers 3, e1039691 (2015). PubMed  PubMed Central  Google Scholar  * Minutoli,

L. et al. Research Article Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood-Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice. Toxicol Sci

148, 311–329 (2015). CAS  PubMed  Google Scholar  * Mruk, D. D. & Cheng, C. Y. The Mammalian Blood-Testis Barrier: Its Biology and Regulation. Endocr Rev 36, 564–591 (2015). CAS  PubMed

  PubMed Central  Google Scholar  * Balasubramanian, S. K. et al. Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration

in rats. Biomaterials 31, 2034–2042 (2010). CAS  PubMed  Google Scholar  * De Jong, W. H. et al. Particle size-dependent organ distribution of gold nanoparticles after intravenous

administration. Biomaterials 29, 1912–1919 (2008). CAS  PubMed  Google Scholar  * Taylor, U. et al. Toxicity of Gold Nanoparticles on Somatic and Reproductive Cells. Adv Exp Med Biol 733,

125–133 (2012). CAS  PubMed  Google Scholar  * Li, W. Q. et al. Gold Nanoparticles Elevate Plasma Testosterone Levels in Male Mice without Affecting Fertility. Small 9, 1708–1714 (2013). CAS

  PubMed  Google Scholar  * Chithrani, B. D., Ghazani, A. A. & Chan, W. C. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 6,

662–668 (2006). ADS  CAS  PubMed  Google Scholar  * Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J. & Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause

acute cytotoxicity. Small 1, 325–327 (2005). CAS  PubMed  Google Scholar  * Jiang, Y. et al. The Interplay of Size and Surface Functionality on the Cellular Uptake of Sub-10 nm Gold

Nanoparticles. ACS Nano 9, 9986–9993 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Cheng, X. et al. Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and

Nonphagocytic Cells in a Size-Dependent Manner. ACS Appl Mater Interfaces 7, 20568–20575 (2015). CAS  PubMed  Google Scholar  * Leclerc, L. et al. Testicular biodistribution of silica-gold

nanoparticles after intramuscular injection in mice. Biomed Microdevices 17, 66 (2015). PubMed  Google Scholar  * Russell, L. D., Bartke, A. & Goh, J. C. Postnatal development of the

Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. Am J Anat 184, 179–189 (1989). CAS 

PubMed  Google Scholar  * Mok, K. W., Mruk, D. D., Lee, W. M. & Cheng, C. Y. A study to assess the assembly of a functional blood-testis barrier in developing rat testes. Spermatogenesis

1, 270–280 (2011). PubMed  PubMed Central  Google Scholar  * Yan, H. H., Mruk, D. D., Lee, W. M. & Cheng, C. Y. Ectoplasmic specialization: a friend or a foe of spermatogenesis?

Bioessays 29, 36–48 (2007). PubMed  PubMed Central  Google Scholar  * Russell, L. D. & Peterson, R. N. Sertoli cell junctions: morphological and functional correlates. Int Rev Cytol 94,

177–211 (1985). CAS  PubMed  Google Scholar  * Chihara, M., Otsuka, S., Ichii, O., Hashimoto, Y. & Kon, Y. Molecular dynamics of the blood-testis barrier components during murine

spermatogenesis. Mol Reprod Dev 77, 630–639 (2010). CAS  PubMed  Google Scholar  * Russell, L. Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am J

Anat 148, 313–328 (1977). CAS  PubMed  Google Scholar  * Mruk, D. D. & Cheng, C. Y. Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the

seminiferous epithelium during spermatogenesis. Endocr Rev 25, 747–806 (2004). CAS  PubMed  Google Scholar  * Hess, R. A. & Renato de Franca, L. Spermatogenesis and cycle of the

seminiferous epithelium. Adv Exp Med Biol 636, 1–15 (2008). PubMed  Google Scholar  * Mok, K. W., Mruk, D. D., Lie, P. P., Lui, W. Y. & Cheng, C. Y. Adjudin, a potential male

contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes. Reproduction 141, 571–580 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Su, L., Cheng,

C. Y. & Mruk, D. D. Adjudin-mediated Sertoli-germ cell junction disassembly affects Sertoli cell barrier function _in vitro_ and _in vivo_. Int J Biochem Cell Biol 42, 1864–1875 (2010).

CAS  PubMed  PubMed Central  Google Scholar  * Kopera, I. A., Su, L., Bilinska, B., Cheng, C. Y. & Mruk, D. D. An _in vivo_ study on adjudin and blood-testis barrier dynamics.

Endocrinology 150, 4724–4733 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Hew, K. W., Heath, G. L., Jiwa, A. H. & Welsh, M. J. Cadmium _in vivo_ causes disruption of tight

junction-associated microfilaments in rat Sertoli cells. Biol Reprod 49, 840–849 (1993). CAS  PubMed  Google Scholar  * Wong, C. H., Mruk, D. D., Lui, W. Y. & Cheng, C. Y. Regulation of

blood-testis barrier dynamics: an _in vivo_ study. J Cell Sci 117, 783–798 (2004). CAS  PubMed  Google Scholar  * Cai, H. et al. Scrotal heat stress causes a transient alteration in tight

junctions and induction of TGF-beta expression. Int J Androl 34, 352–362 (2011). PubMed  Google Scholar  * Xia, W., Wong, E. W., Mruk, D. D. & Cheng, C. Y. TGF-beta3 and TNFalpha perturb

blood-testis barrier (BTB) dynamics by accelerating the clathrin-mediated endocytosis of integral membrane proteins: a new concept of BTB regulation during spermatogenesis. Dev Biol 327,

48–61 (2009). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant No: 31500817 and 81430027),

the National Basic Research Program (973 Program) (No: 2014CB943100 to F.S.), the Fundamental Research Funds for the Central Universities (to R.H.), and scientific research of BSKY

(XJ201412), 2015xkj002 (to W.Q.L.) from Anhui Medical University. AUTHOR INFORMATION Author notes * Lin Yu and He Rong contributed equally to this work. AUTHORS AND AFFILIATIONS * School of

Life Sciences, University of Science and Technology of China, Hefei, 230027, Anhui, P.R. China Yu Lin & Fei Sun * Department of Chemical Physics, Hefei National Laboratory for Physical

Sciences at the Microscale, Center of Advanced Nanocatalysis (CAN-USTC), University of Science and Technology of China, Hefei, 230026, Anhui, P.R. China Rong He & Yushan Yang *

Department of Pathophysiology, Basic Medical College, Anhui Medical University, Hefei, 230032, Anhui, P.R. China Liping Sun & Wenqing Li Authors * Yu Lin View author publications You can

also search for this author inPubMed Google Scholar * Rong He View author publications You can also search for this author inPubMed Google Scholar * Liping Sun View author publications You

can also search for this author inPubMed Google Scholar * Yushan Yang View author publications You can also search for this author inPubMed Google Scholar * Wenqing Li View author

publications You can also search for this author inPubMed Google Scholar * Fei Sun View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS F.S. and

W.Q.L. designed the studies and wrote the paper. Y.L., R.H. and W.Q.L., L.P.S., and Y.S.Y. performed the experiments. Y.L., R.H. and W.Q.L., performed related data analysis. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND PERMISSIONS This work is

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al._ Pentacle gold–copper alloy nanocrystals: a new system for entering male germ cells _in vitro_ and _in vivo_. _Sci Rep_ 6, 39592 (2016). https://doi.org/10.1038/srep39592 Download

citation * Received: 30 March 2016 * Accepted: 24 November 2016 * Published: 21 December 2016 * DOI: https://doi.org/10.1038/srep39592 SHARE THIS ARTICLE Anyone you share the following link

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