Endoplasmic reticulum stress response in the spermatogenic cultures isolated from non-obstructive azoospermic patients with spermatogenic arrest
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Department of 2Medical Biology, İstanbul University-Cerrahpaşa, Faculty of Medicine, İstanbul, Turkey
Department of 3Medical Biology and Genetics, İstanbul Aydın University, Faculty of Medicine, İstanbul, Turkey
Department of 4Histology and Embryology, Biruni University, Faculty of Medicine, İstanbul, Turkey
Department of 5Histology and Embryology, Altınbaş University, Medical Faculty, İstanbul, Turkey
ABSTRACT
Background and Aims: Cells activate a defense mechanism calledendoplasmic reticulum stress response when the amount of unfolded proteins exceedsfolding capacity of endoplasmic reticulum, which induces expression of heat shock proteins. As a member of heat shock protein (HSP) 70 family, HSPA5/BiP/GRP78 hasbeen identifed in mammalian spermatozoa and expressed in cytoplasm of humanspermatocytes and round spermatids. However, the role of HSPA5 in spermatogenicarrest testis remains to be determined. Thus, we aimed to reveal HSPA5 immunoreactivity in the isolated mixed cell clusters from non-obstructive azospermic male byusing immunocytochemical techniques and to evaluate the success of in vitro spermiogenesis. Materials and Methods: Spermatogenic cells were isolated from testicularbiopsies of non-obstructive azospermic patients (n=18) diagnosed with maturation arrest, immunostained with HSPA5 antibody, cell suspension was cultured with G-in-vitrofertilization culture medium supplemented with 25 IU/L recombinant follicle stimulatinghormone and 1 µmol/L testosterone for six days. Cultured cells were analyzed by lightand electron microscopical techniques. Results: The rate of success by in-vitro spermiogenesis was 16.7%. Immunocytochemical analysis revealed that HSPA5 scoresof all cells were signifcantly higher than control group (p<0.05) and that of spermatogenic cells was signifcantly higher than non-spermatogenic cells (p<0.0001). Someof differentiated spermatids lost their ultrastructural morphology, underwent apoptosisprobably due to endoplasmic reticulum stress. Conclusions: Low success of in vitrospermiogenesis may be due to induced endoplasmic reticulum stress even in an idealculture conditions acquired by follicle-stimulating hormone and testosterone. Eliminating endoplasmic reticulum stress during in vitro spermiogenesis is promising in treatment of spermatogenic arrest.
INTRODUCTION
Male infertility has been reported to affect approximately 7% of the male population, and nearly 1%of infertile men are azoospermic (1). One of thecause of azoospermia is the spermatogenic arrest,the pause of spermatogenesis in some seminiferous tubules during the formation phase of spermatocytes or spermatids. Spermatogenic arresthas been diagnosed by testicular biopsy in menwho had either severe oligospermia (partial arrest)or azoospermia (complete arrest), normal testicular volume, and depending on the etiology normal,high, or low levels of gonadotropins. The radiotherapy, heat, and chemotherapy have also beenreported to cause an arrest in spermatogenesisdirectly or indirectly. Irreversible arrest at primary spermatocyte or spermatid level have a geneticorigin due to chromosomes anomalies either in somatic cells or in germ cells (2).In assisted reproductive techniques, microsurgicaltesticular sperm extraction (m-TESE) and intra-cytoplasmic sperm injection (ICSI) do not help theazoospermic patients with a complete spermatogenic arrest. However, in vitro differentiation ofdiploid germ cells to mature haploid germ cell hasthe potential to benefit these patients (1). Severalexperimental tests and approaches have been developed using whole testis tissue or isolated singlecells from testis biopsies in two-dimensional (2D)or three-dimensional cell culture systems (3D) toinvestigate the reasons of spermatogenic arrest(3-5). Recent advances of in mammalian models ofvitro spermatogenesis are promising in response toovercome the spermatogenic arrest in the humanclinical setting.There is a highly demand to develop an effectiveculture technique by which haploid productive andfunctional spermatozoa could be produced fromdiploid germ cells isolated from the azoospermicpatients (6). If a successful in-vitro spermatogenesis culture method could be improved, in-vitro fertilization (IVF) treatment for patients with non-obstructive azoospermia (NOA) enable physicians toovercome the spermatogenic arrest (7). Thus, thereis an urgent need for an efficient method for in-vitrospermatogenesis. One of these methods, isolatedcell suspension cultures are widely used and preferred to investigate the pathological mechanismsof spermatogenic arrest (6). In order to improvethe micro environment for in vitro differentiationof isolated cells, mediums supplemented with follicle stimulating hormone (FSH) and testosteronewere used for the differentiation of spermatid intoelongated spermatid, implicating the critical roleof FSH and testosterone in spermatogenesis (6).Molecular chaperones are able to confer cellularresistance to environmental stressors, and the majority of these chaperone families are related withthe cell stress response or, more commonly knownas heat shock proteins (HSPs) (8). Molecular chaperones also participate in a number of normalcellular functions, including metabolism, growth,differentiation and apoptosis (9). The regulatorsof HSPs, heat shock transcription factors (HSFs),are well known for their cytoprotective functionsduring cellular stress but less known for potentialroles in gametogenesis. All HSF family membersare expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate tomaintain proper spermatogenesis (10).In eukaryotic cells, proteins synthesized in theendoplasmic reticulum (ER) are properly foldedwith the assistance of HSPs. When the amountof unfolded proteins exceeds the folding capacity of the ER, cells activate a defense mechanismcalled the ER stress response (unfolded proteinresponse-UPR), which induces expression of ERchaperones and transiently attenuates proteinsynthesis to decrease the burden on the ER (11).Along with its role in protein folding, HSPA5/BiP/GRP78 (heat shock 70kDa protein 5a- major ERchaperone) is also known to be a key component in modulating the UPR. In certain severe conditionsof ER stress, however, the protective mechanismsactivated by the UPR are not sufficient to restorenormal ER function and cells die by apoptosis (12,13). HSPA5 is a member of the HSP70 family thathas been identified in mammalian spermatozoaand expressed in the cytoplasm of human spermatocytes and round spermatids. Recent evidencesuggests that HSPA5 may play important role(s)in the function of Sertoli cells, mature human andmouse spermatozoa (11,13-15). However, the roleof HSPA5 in spermatogenic arrest testis remainsto be determined. Thus, we aimed to reveal theHSPA5 immunoreactivity in the isolated mixedcell suspensions collected from TESE biopsies ofNOA patients with spermatogenic arrest by usingimmunocytochemical techniques and to evaluatethe success of in vitro spermiogenesis by light andtransmission electron microscopy.
MATERIALS
Patients
The protocol for establishing primary human cellcultures from testicular biopsies obtained duringTESE was approved by the Ethics Committee ofClinical Research Center of Cerrahpasa School ofMedicine, Istanbul University, Istanbul, Turkey(Date: 3rd August 2010, Approval number: 23396).This study was managed in accordance with theprinciples of the Declaration of Helsinki (as revisedin Brazil in 2013), the International Conference onHarmonization guidelines for Good Clinical Practice and was issued legal approval by the TurkishHealth Ministry. The signed written informed consent was obtained from all patients prior to thestudy.Testicular biopsies of NOA patients (n=18) diagnosed with maturation arrest following TESEprocedures were selected for the study. Exclusioncriteria included the diagnoses of pre-testicularazoospermia, post-testicular azoospermia, obstructive azoospermia and Sertoli cell-only patterns. All testis biopsies were dissected mechanically byhypodermal needles immediately after examination for any spermatozoa in sperm preparation medium (Medicult) conditioned by 5% CO2 under aninverted microscope (Olympus IX71). The biopsypieces was centrifuged and washed at 1200 rpm for10 min inside the same medium. The supernatantcontaining cell suspension was divided for the fourstages of experiments, namely light microscopy,immunocytochemistry, and cell culture and transmission electron microscopy.
Light Microscopy
To isolate spermatogenic cells, discontinuous density gradients of 40% and 80% were prepared bysperm separation medium (Supra Sperm, Medicult) and sperm preparation medium (Medicult).1ml of cell suspension was placed on the gradientsand centrifuged at 2000 rpm for 20 min. Separatedbands of supernatant were divided into tubes andall cell suspension from 40% and 80% gradientswere counted by using a Makler counting chamber(Self-Medical Instruments ltd.) under a light microscope (Nicon).The cell suspensions were smeared on eight slides,and fixed by methanol solution of ready-to-use set(Hemadiff MGG, GBL) for 10 min. Four of slideswere stained by May-Grünwald Giemsa (HemadiffMGG, GBL) for 30 seconds and washed under running water and dried. The stained slides were examined by 100x immersion objective under a lightmicroscope (Olympus BX 61) and photographed bya digital camera attached to microscope (OlympusDP 72). The remaining slides were used in immunocytochemical methods.
Immunocytochemical Analysis
Indirect immunoperoxidase technique was usedfor detection of HSPA5 protein on smears prepared from cell suspension from both gradients.The immunocytochemistry procedures were performed according to our previous study (16). For blocking the endogenous peroxidase, 3% hydrogenperoxidase prepared by methanol and distilled water was used. 5% normal goat serum (Vector laboratories) was used to prevent non-specific binding.As a primary antibody, anti-BiP/GRP78 (SigmaAldrich) was used as diluted for 1:1000. As a secondary antibody, biotinylated goat anti-rabbit antibody (Vector) was used. Avidin-biotin-peroxidasekit (LabVision) was used for the formation of antigen-antibody complex. 3,3-diaminobenzidine tetrahydrochloride dehydrate (DAB, LabVision) wasused as a chromogen. Mayer’s Hematoxylin wasused for counterstaining. Negative control slideswere not marked with primary antibody and positive control slide included the control cells (erthyrocytes with no nuclei).Labeled cells were assessed by two researchersand photographed by using a camera-attachedlight microscope (Olympus BX 61). The intensityof HSPA5 immunostaining was semi-quantitatively evaluated among spermatogenic cells (mostlyspermatids) and non-spermatogenic (mostly Sertoli) cells by using H-SCORE analysis (17).
Cell Culture
0.5 ml suspension from 40% gradient includingapproximately 1 x 106 cells was washed and incubated with G-IVF culture medium (Vitrolife), andsupplemented with 25 IU/L rFSH (MBL international corporation) and 1µmol/L testosterone (Nebido-BAYER) for six days in an incubator airedwith 5% CO2 at 35 °C. The mediums were changedonce in two days. The doses of rFSH and testosterone were selected according to the literature (18).Pre-cultured and post-cultured cells were photographed under an inverted microscope (OlympusIX71). Matured spermatids were counted and noted.Post-cultured cells were centrifuged at 2000 rpmfor 10 min, smeared and stained with May-Grünwald Giemsa (Hemadiff MGG, GBL), and photographed.
Transmission Electron Microscopy
The cell suspension collected from 40% gradientwere centrifuged at 1200 rpm for 10 min in spermwashing medium and the supernatant was expelled.The pellet was immersion fixed at 4° C for 4 h in4% glutaraldehyde (Merck Millipore, USA) solutionprepared in 0.1 M PBS (pH = 7.3). Following washing in a Milloning phosphate buffer, the pellet waspostfixed in 1% Osmium tetroxide (EMS Diasum,USA) for 30 min. The pellet was washed again withthe buffer and immersed in 2% Agar solution. Solidified tissues in Agar were dissected into 1 mm3blocks, washed with the buffer and dehydrated byimmersion in grading series of alcohol (10%, 30%,50%, 70%, 80%, 96%, 100%). After application ofpropylene oxide, the sample was embedded in 1:1propylene oxide + araldite mixture, 1:3 propyleneoxide + araldite mixture and lastly pure araldite,respectively. Polymerization was performed at 60°C for 48 hours. Araldite blocks were cut in 60-70mm thickness by using an ultramicrotome (ReichertUM3) and thin-sections were placed on copper gridsand stained with saturated uranyl acetate and counterstained with Reynold’s lead citrate. Contrastedgrids were examined by a transmission electron microscope (Jeol JEM 1001 TEM) and photographedan imaging system software (Jeol Mega View III).
Statistical Analysis
Statistical analyses of the data were performed using GraphPad InStat Software (Version 3.06). Results are presented as mean ± SEM and comparedby Paired t test. p<0.0001 was considered significant. For post-hoc multiple comparison, DunnMultiple Comparison test was used and p<0.05was considered significant.
RESULTS
Pre-culture Findings
According to the histomorphometric counting, thepercentage of round cells (potential Sa spermatids)isolated from 40% gradient was significantly higherthan the percentage of round cells from 80% gradient (p<0.05). However, the percentage of non-spermatidcells from 80% gradient was distinctly higher thanthat cells from 40% gradient (P<0.0001) (Table 1).Immunocytochemical analysis revealed thatH-SCORE for HSPA5 was significantly higher inall types of cells than the control group (p<0.05). H-SCORE of spermatogenic cells was significantlyhigher than the scores of non-spermatogenic cells inboth 40% and 80% gradients (p<0.0001). However,there was no significant difference between the immunostaining of same non-cultured cell types isolated from 40% and 80% gradients (Table 2, Figure 1).
Post-culture Findings
Round (Sa) and elongating spermatids (Sb) wereobserved in the cultures before the incubation.After the very first days of incubation with rFSHand testosterone containing medium, round (Sa)spermatids and elongated spermatids (Sd) wereobserved in the cultures. For 7 days, every examination of cultures showed elongated spermatidsin three samples isolated from testicular biopsies(Figure 2). As a result, in-vitro spermiogenesis wassuccessful in only 3 (16.7%) of 18 cultured samplesup to elongated spermatid stage (Figure 3).
Ultrastructural Findings
When isolated cells before the culture were examined under TEM, round spermatids (Sa) with condensing nuclei were observed, as parallel to light microscopic results. However, some spermatidshad damaged morphology, degenerated membraneand acrosomes, and apoptotic cytoplasms. Nucleihad also apoptotic bodies (Figure 4). Connectivetissue cells and collagen bundles and elastic fiberswere observed in some samples. Post-culture TEM micrographs occasionallyshowed round spermatids (Sa) and elongated spermatids (Sd) but most of spermatids were apoptoticor degenerated (Figure 5).
DISCUSSION
This work demonstrates that the protein amountof HSPA5 in spermatogenic cells (specifically spermatids) was higher than the amount in non-spermatogenic cells which were isolated from TESE biopsies of NOA patients with spermatogenic arrest,by using a density gradient method. Moreover,40% gradient was successful to separate roundspermatogenic cells from the other cells comparedwith 80% gradient. More importantly, our post-culture results demonstrated that in vitro maturation of isolated round spermatids, which were arrested during spermatogenic differentiation, wasachieved by using rFSH and testosterone, by a success ratio of 16.7%. However, the isolation methodsand culture conditions led to the ultrastructuraldegenerations in cells and even cellular apoptosis.Spermatogenic arrest can occur due to a gonadotropin insufficiency or following a germ cell damage after the chemotherapy or radiotherapy. Thearrest is most frequently observed at primaryspermatocyte level and less frequently at spermatid level. Reversible arrest may be due to consecutive hormonal, thermic, or toxic factors and can beresolved spontaneously or by a specific treatment.Spermatogenic arrest at spermatid level is usuallydue to genetic factors resulting in irreversible azoospermia (2). Irriversible azoospermia can be overcome by reorganization of testicular cells isolatedfrom TESE biopsies obtained from men enrolled ina standard clinical assisted reproduction program.The cell clusters can be cultivated with somaticcell types that are essential to support spermatogenesis for at least 3 months (2,3,6). A perfectculture model would include the combination ofsomatic and germ cells which mimics the seminiferous epithelium, for the maintenance of the spermatogonial stem cell and suitable equilibrium ofself-renewal and differentiation in the pre-meioticphase of spermatogenesis (6). However, the levelof spermatogenic arrest is very crucial to understand the reason of arrest and to find a treatment.The endocrine hormones also play pivotal roles inthe pathophysiology of this process. In order toimprove the micro environment for in vitro differentiation of isolated cells, the effect of hormones,growth factors and feeder cells were investigatedon spermatids by Movahedin et al. (19). Mousespermatid were cultured in DMEM with FBS andsupplemented with FSH, testosterone and co-culture (Feeder cell), resulted in the differentiation ofspermatid into elongated spermatid at the 2nd dayof culture in the hormones supplemented group. In2010, Xie et al. cultured spermatogonia and Sertoli cells from immature buffalo testes with FBSbased media supplemented with FSH, testosteroneand retinoic acid (20). Spermatid-like cells with aflagellum were observed after 30 days of culture,suggesting the critical role of FSH and testosterone in spermatogenesis. Thus, we used rFSH andtestosterone to improve in vitro spermiogenesis forthe biopsies collected from the NOA patients withspermatogenic arrest and the spermatids of 16.7%of the samples succeeded to differentiate into elongated spermatid.Culture conditions had promising effects on in vitrospermatogenesis by reducing the number of apoptotic germ cells (21). It was reported that the human round spermatids in co-culture with humanfibroblast as feeder cells for up to 5 days experienced a spermiogenesis (22), while spermatogoniaand spermatocytes co-cultured with Sertoli cellsin a supplemented culture with testosterone andFSH differentiated into late spermatids (23). Iwanami et al. observed the differentiation of type-Aspermatogonia of an immature (7-day-old) rat into spermatid when these cells were co-culturedwith Sertoli cells, however, the resulted spermatidwas not fertile (24). Menegazzo et al. has recommended that porcine fetal Sertoli cells are properto indorse the development of human spermatidsby long-term in vitro co-culture (25). However, theproduction of haploid cells for fertilization in thepresence of feeder cells or somatic cells also raisesthe problem of in vitro contamination risk and anepigenetical impact on the health of any offspring.Recently, the use of biocompatible scaffolds is another effort to improve the efficiency of in vitrospermatogenesis (26). Lee et al. cultured the isolated testicular cells from immature rats on biodegradable poly scaffolds and after 18 days of culture, 65% of cells were successfully attached thescaffolds with 75% viability. The differentiationrate of germ cells was also higher compared to cellsseeded on a monolayer (26). In another study, human isolated spermatogonial stem cells from NOApatients were cultured in a media supplementedwith knockout serum replacement, resulted in apromoted differentiation (27). Wang et al. reported the successful generation of haploid spermatidfrom mouse spermatogonial stem cells when theycultured the isolated cells in 10% FBS supplemented media for 3 days, subsequently treated the cellswith medium enriched with retinoic acid for differentiation (28). However, the efficiency of haploidcells production was low. Thus, we used an isolatedcell suspension culture to investigate the effects ofFSH and testosterone on in vitro spermiogenesis ofround spermatids isolated from biopsies of patientswith spermatogenic arrest but we did not checkthe efficiency and the fertility of haploid producedcells. Although the ultrastructure of post-culturedcells gave a valuable information about the statusof spermatogenic cells, there is still need to investigate the molecular organization and functionalityof these differentiated cells by further molecularanalysis.HSPA5, formerly known as GRP78/BiP is a member of the HSP70 family identified in mammalianspermatozoa. HSPA5 commonly localized in ERlumen plays a critical role in protein transport, folding and assembly (29). As unfolded proteins accumulate in the ER lumen, HSPA5 disassociatesfrom several stress sensors, enabling the proteinfolding while simultaneously promoting the activation of the released sensors and the initiationof ER-stress signaling pathways (29). It was reported that Hspa5 mRNA expression was rapidlyincreased in Sertoli cells following an exposure toenvironment contaminants linked to the disruption of testicular development and decrease malefertility (14). However, in addition to this protective role, HSPA5 may also play crucial role in thefunction of mature human and mouse spermatozoasince it has been identified on the surface and cytoplasm of human spermatozoa and round spermatids (11, 13, 30). However, the role of human spermsurface HSPA5 remains to be determined for NOApatients with spermatogenic arrest. In the present study, immunocytochemical analysis revealedthat HSPA5 was significantly elevated especially in spermatogenic cells, suggesting induced ERstress is in pre-cultured cells. It is a controversialissue that mechanical dissection of the seminiferous tubules of azoospermic tissues and cell isolation procedures for in-vitro maturation studies canbe hazardous for cell homeostasis. TEM resultsof this study confirmed the degenerative effects of isolation method on the spermatogenic cells.The mechanical forces raised from centrifugationcannot be rule out from the increased ER stresssince spermatogenic cells are more delicate andless protected in culture conditions compared tothe somatic (non-spermatogenic) cells. Low culturesuccess may be another result of induced ER stressin spermatogenic cells. However, it is noteworthythat the application of ideal culture conditionswith rFSH and testosterone conditioned mediumobviously supports in vitro spermiogenesis.The findings of this study lend support to the concept that the rough environment may modulatethe formation of functional chaperone complexesduring in vitro spermiogenesis and that alternations in the physiological conditions of spermatogenic cells may result in ER stress, resulting inultrastructural changes and apoptosis. Thesefindings may provide the cellular basis for advances in human in vitro spermatogenesis and/or thepossibility for maturation of spermatids within anatural physiological environment. More intensive struggles and works are needed to develop themost optimal culture conditions for in vitro spermiogenesis, and to understand the effects of usingthe endocrine factors in assisted reproductive techniques.
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