Coincident Phosphatidic Acid Interaction Restrains Drp1 in Mitochondrial Division.

Mitochondria divide to control their size, distribution, turnover, and function. Dynamin-related protein 1 (Drp1) is a critical mechanochemical GTPase that drives constriction during mitochondrial division. It is generally believed that mitochondrial division is regulated during recruitment of Drp1 to mitochondria and its oligomerization into a division apparatus. Here, we report an unforeseen mechanism that regulates mitochondrial division by coincident interactions of Drp1 with the head group and acyl chains of phospholipids. Drp1 recognizes the head group of phosphatidic acid (PA) and two saturated acyl chains of another phospholipid by penetrating into the hydrophobic core of the membrane. The dual phospholipid interactions restrain Drp1 via inhibition of oligomerization-stimulated GTP hydrolysis that promotes membrane constriction. Moreover, a PA-producing phospholipase, MitoPLD, binds Drp1, creating a PA-rich microenvironment in the vicinity of a division apparatus. Thus, PA controls the activation of Drp1 after the formation of the division apparatus.

Effect of platelet rich plasma versus melatonin on testicular injury induced by Busulfan in adult albino rats: a histological and immunohistochemical study.

This study was done to estimate the testicular histological alterations induced by Busulfan (BUS) and compare the possible protective effects of melatonin (MT) and platelet rich plasma (PRP) in a rat model. Sixty-four male rats were dispersed into: control group, BUS group, melatonin group, and PRP group. Blood samples were processed for biochemical analysis. Tissue specimens were managed for light and electron microscopic studies. Immunohistochemical expression of vimentin and proliferating cell nuclear antigen (PCNA) was performed. Busulfan induced severe testicular damage in all studied methodologies. It showed a statistically significant decrease in serum testosterone and elevation of MDA when compared to the control group. Abnormal testicular cytostructures suggesting defective spermatogenesis were observed: distorted seminiferous tubules, deformed spermatogenic cells, low germinal epithelium height, few mature spermatozoa, and also deformed barrier. Vimentin and PCNA expressions were reduced. Ultrastructurally, Sertoli cells and the blood testis barrier were deformed, spermatogenic cells were affected, and mature spermatozoa were few and showed abnormal structure. Both melatonin and PRP induced improvement in all the previous parameters and restoration of spermatogenesis as confirmed by improvement of Johnsen's score from 2.6 ± .74 to 7.6 ± .92. In conclusion, melatonin and PRP have equal potential to ameliorate the testicular toxicity of BUS. Melatonin can provide a better noninvasive way to combat BUS induced testicular injury.

A Potential Role for the Gsdf-eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor ß-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor ß) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

Photoperiod dependent expression of estrogen receptor alpha in testes of Japanese quail: Involvement of Withania somnifera in apoptosis amelioration.

Light plays important function in the regulation of reproduction in vertebrates including birds. The prolonged long day length exposure causes reproductively inactive state or photorefractoriness in many avian species including Japanese quail. Withania somnifera (WS) is a medicinal plant known to have beneficial effects on stress and infertility. The study investigates the physiological effect of WS on the light-induced stress in quail mediated by estrogen receptor alpha. Quails were exposed to long day length for three months and then transferred into intermediate day length to make them photorefractory (PR) while controls under natural day length. Administration of Withania somnifera root extract (WSRE) in PR quail induces estrogen and decreases corticosterone in male Japanese quail. Immunoreactivity of ERα decreased in testis of PR quail and increased after oral administration of WSRE compared to control. Expression of ir-Caspase-3 and ir-p53 in the testis increased in PR while decreased in PR + WS. Histologically, seminiferous tubules size decreased in PR whereas increased in PR + WS quails. Scanning electron microscopic study reveals sperms in clusters with proper head and tail in control. In PR quails sperms were few and distorted while WSRE improved the sperm morphology. From the study, it is concluded that during photorefractoriness gonadal regression occurs due to testicular apoptosis which causes stress. WSRE helps to overcome stress and improve reproductive performance via increase in expression of ir-ERα during PR condition. Further, the stress ameliorating effect of WSRE in reducing apoptosis mediated by ir-Caspase-3 and ir-p53 in the testes is clearly evident in Japanese quail.

Genome-wide identification and functional analysis of long non-coding RNAs and mRNAs in male mice testes at the onset of puberty after low dose lead exposure.

Many researchers have studied the relationship between lead (Pb) and testis injury, but the underlying mechanisms are still unknown. The participation of long non-coding RNAs (lncRNAs) in biological processes has been proposed. To comprehensively gain insight into the molecular toxicity of Pb, expression patterns are analysed through RNA sequencing (RNA-seq) in male mice treated with 200 mg/L of Pb through the drinking water for 90 days at the onset of puberty. A total of 614 differentially expressed (DE) lncRNAs were included (p ≤ 0.05 and fold change ≥2), of which 288 were up-regulated, and 326 were down-regulated. A total of 2295 DE mRNAs (p ≤ 0.05 and fold change ≥2), including 1202 up-regulated and 1093 down-regulated ones, were found in the testes of Pb-exposed group. Functional analysis results showed that several lncRNAs might be implicated in the bio-pathway of mitogen-activated protein kinase (MAPK) signaling pathway. Finally, seven pairs of lncRNA-mRNA co-expression were established in mice testes and confirmed by RT-qPCR. Moreover, the DE genes were also altered in Sertoli cells. Therefore, our research might be helpful for future exploring the effects of Pb exposure on lncRNA in testis, as well as its function.

ATP8a1, an IFT27 binding partner, is dispensable for spermatogenesis and male fertility.

Intraflagellar transport 27 (IFT27) is a key regulator for spermiogenesis and male fertility in mice. ATP8a1, a protein involved in the translocation of phosphatidylserine and phosphatidylethanolamine across lipid bilayers, is the strongest binding partner of IFT27. To investigate the role of ATP8a1 in spermatogenesis and male fertility, the global Atp8a1 knockout mice were analyzed. All mutant mice were fertile, and sperm count and motility were comparable to the control mice. Examination of testis and epididymis by hematoxylin and eosin staining did not reveal major histologic defects. These observations demonstrate that ATP8a1 is not a major spermatogenesis regulator. Given that a tissue-specific paralogue of ATP8a1, ATP8a2, is present, further studies with double-knockout models are warranted to delineate any compensatory functions of the two proteins.

Chronic unpredictable stress disturbs the blood-testis barrier affecting sperm parameters in mice.

RESEARCH QUESTION: Does chronic stress affect the key proteins and sperm parameters of the blood-testis barrier (BTB)? DESIGN: C57Bl/6 mice were divided into two groups: a non-treated control group and a chronic unpredictable stress (CUS) applied group. The stress status of the animals was confirmed with behavioural tests. Histopathologic evaluation was conducted by haematoxylin and eosin staining and electron microscope. Malondialdehyde, corticosterone and testosterone levels were evaluated in peripheral blood. Expression levels of BTB proteins, namely zonula occludens-1 (ZO-1), claudin-11 (CLDN11) and clathrin in Sertoli cells, were assessed by Western blotting and immunofluorescence techniques. Sperm samples were collected from cauda epididymis, and sperm parameters analysed. RESULTS: The stress model was confirmed by behavioural tests. Histopathological evaluation of the testes demonstrated a mild degeneration in seminiferous tubules. Malondialdehyde (P = 0.008) and corticosterone levels increased (P = 0.004) and testosterone levels decreased (P = 0.005) in the CUS group. Electron microscopic evaluation confirmed the damage in BTB integrity in the CUS group. Western blot analysis showed that ZO-1 and CLDN11 levels were significantly decreased, although clathrin levels were unchanged. Although sperm concentration and total motility rate were not significantly different between the groups, progressive motility (P = 0.03), normal sperm morphology (P = 0.04), chromatin integrity (toluidine blue) (P = 0.002) and the acrosomal reaction rate (P = 0.002) were significantly decreased, and acrosomal abnormality rate was dramatically increased (P = 0.04) in the CUS group. CONCLUSIONS: In mice, CUS disrupted BTB integrity and impaired sperm parameters. A decrease in ZO-1 and CLDN11 expression levels may be proposed as the causative factor.

Cytogenetic and testicular histological alterations induced by sulphur dioxide in dried apricot leather.

Sulphur dioxide (SO2) is used as a preservative in food to prevent its discolouration, and to inhibit the growth of bacteria. Little data is available concerning its in vivo hazardous impact.The present study is therefore designed to examine the cyto-genotoxic potential and the testicular histological alterations in adult mice, induced by SO2 present in the dried apricot leather used to prepare the oriental drink Qamar Al-Deen. Two different forms of drinks were tested; cold and boiled drinks. Animals were placed into 4 groups. The first group received distilled water as a negative control.The second and third groups received orally the drink for 28 days in the form of a cold and a boiled drink, respectively. Animals of the fourth group received cyclophosphamide, they were used as a positive control for cyto-genotoxic tests. The chromosomal aberrations, as well as sperm abnormalities, were significantly elevated in animals that received the two different drink preparations. The mitotic index significantly decreased in comparison with negative and positive controls. Furthermore, histological examination showed different degrees of alterations in the testis. Our results suggest that the presence of SO2 inside the apricot leather might be responsible for these changes. Thus, these remarkable hazardous effects of SO2 on male albino mice could be used as a potential guide for the prediction of its human health impact. Furthermore, consumers could be advised to prevent excessive consumption of the drink (Qamar Al-Deen) prepared from dried apricot leather.

Inhibition of endoplasmic reticulum stress-related autophagy attenuates MCLR-induced apoptosis in zebrafish testis and mouse TM4 cells.

Microcystin-leucine arginine (MCLR), a widespread environmental contaminant produced by cyanobacteria, poses a severe threat to the male reproductive system. However, the mechanisms of MCLR-induced testis injury accompanied by autophagy are still obscure. This study aimed to investigate the effects of MCLR on autophagy and apoptosis on the male reproductive system and its mechanism both in vitro and in vivo. MCLR caused damage to the testis of zebrafish, resulting in decreased hatching and growth retardation in the offspring. It also remarkably enhanced autophagic flux by elevating the expression of LC3BII, ATG5, and ATG12 proteins. The autophagic flux was also confirmed through the formation of autophagosomes in the ultrastructure of the zebrafish testis and the accumulation of LC3-positive puncta in zebrafish testis and mouse TM4 cells. Further evaluations revealed that inhibition of autophagy by 3-methyladenine (3-MA) significantly attenuated MCLR-induced apoptosis. This finding indicated that autophagy plays an essential role in cell death in the male reproductive system. Besides, inhibiting endoplasmic reticulum (ER) stress using 4-phenylbutyrate (4-PBA) remarkably blocked autophagy and partially suppressed apoptosis in TM4 cells induced by MCLR. This phenomenon suggested that ER stress-related autophagy was involved in MCLR-induced apoptosis. This study reveals crosstalk between ER stress and autophagy via the PERK/eIF2α/ATF4 signaling pathway. It further suggests that ER stress-related autophagy contributes to MCLR-induced apoptosis and injury in the male reproductive system. These findings provide a novel insight into MCLR-induced impairments of the testis.

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

Nickel oxide nanoparticles induce genotoxicity and cellular alterations in the ground beetle Blaps polycresta (Coleoptera: Tenebrionidae).

Nickel nanoparticles (Ni-NPs) have advantageous applications in the industry; however, little is known of their adverse effects on biological tissues. In the present study, the ground beetle Blaps polycresta was employed as a sensitive indicator for nickel oxide nanoparticles (NiO-NPs) toxicity. Adult male beetles were injected with six dose levels of NiO-NPs (0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg/g body weight). Mortality was reported daily over 30 days under laboratory conditions to establish an LD50. Nickel was detected in the testicular tissues of the beetles using X-ray analysis and transmission electronic microscopy. Beetles treated with the sublethal dose of 0.02 mg/g were selected to observe molecular, cellular, and subcellular changes. Gene transcripts of HSP70, HSP90, and MT1 were found to be increased >2.5-, 1.5-, and 2-fold, respectively, in the treated group compared with the controls. Decreased gene expression of AcPC01, AcPC02, and AcPC04 (≤1.5-, ≤2-, and < 2.5-fold, respectively, vs. controls) also were reported in the treated group. Under light microscopy, various structural changes were observed in the testicular tissues of the treated beetles. Ultrastructure observations using scanning and transmission electron microscopy showed severe damage to the subcellular organelles as well as deformities of the heads and flagella of the spermatozoa. Therefore, the present study postulated the impact of NiO-NPs in an ecological model.

Bisphenol A decreases expression of Insulin-like factor 3 and induces histopathological changes in the Testes of Rats.

Bisphenol A (BPA) is a chemical agent known to have detrimental reproductive and developmental effects. The tissue-specific impacts of BPA exposures and target tissues sensitiveness to BPA are still unclear. The aim of this study was to determine the short- and long-term dose-dependent toxic effects of BPA on rat testes. Forty-eight Wistar albino male rats were divided into four groups each containing 12 rats. To induce toxicity, BPA was administered orally at three different dosages (50, 100, and 200 mg/kg) for 14 and 28 days, respectively. Testis tissues were examined using light and electron microscopy, immunohistochemistry, and biochemical methods. Serum testosterone (T) and luteinizing hormone (LH) levels were measured. Additionally, insulin-like factor 3 (INSL3) as a marker of Leydig cell function was evaluated immunohistochemically. Groups administered high doses of BPA showed severe degenerations such as testicular atrophy, spermatogenic arrest, and interstitial edema in testis. Also, a significant decrease in INSL3 immunoreactivity and serum LH and T levels was found. The results indicated that both increased exposure time and dosage of BPA caused more serious detrimental effects on testes in the rat. Decreased INSL3 and T levels was evidence of Leydig cell function impairment due to BPA.

Six Decades of Research on Human Fetal Gonadal Steroids.

Human fetal gonads acquire endocrine steroidogenic capabilities early during their differentiation. Genetic studies show that this endocrine function plays a central role in the sexually dimorphic development of the external genitalia during fetal development. When this endocrine function is dysregulated, congenital malformations and pathologies are the result. In this review, we explain how the current knowledge of steroidogenesis in human fetal gonads has benefited from both the technological advances in steroid measurements and the assembly of detailed knowledge of steroidogenesis machinery and its expression in human fetal gonads. We summarise how the conversion of radiolabelled steroid precursors, antibody-based assays, mass spectrometry, ultrastructural studies, and the in situ labelling of proteins and mRNA have all provided complementary information. In this review, our discussion goes beyond the debate on recommendations concerning the best choice between the different available technologies, and their degrees of reproducibility and sensitivity. The available technologies and techniques can be used for different purposes and, as long as all quality controls are rigorously employed, the question is how to maximise the generation of robust, reproducible data on steroid hormones and their crucial roles in human fetal development and subsequent functions.

The essential role of intraflagellar transport protein IFT81 in male mice spermiogenesis and fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism that is indispensable for the formation and maintenance of cilia and flagella; however, the implications and functions of IFT81 remain unknown. In this study, we disrupted IFT81 expression in male germ cells starting from the spermatocyte stage. As a result, homozygous mutant males were completely infertile and displayed abnormal sperm parameters. In addition to oligozoospermia, spermatozoa presented dysmorphic and nonfunctional flagella. Histological examination of testes from homozygous mutant mice revealed abnormal spermiogenesis associated with sloughing of germ cells and the presence of numerous multinucleated giant germ cells (symblasts) in the lumen of seminiferous tubules and epididymis. Moreover, only few elongated spermatids and spermatozoa were seen in analyzed cross sections. Transmission electron microscopy showed a complete disorganization of the axoneme and para-axonemal structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers. In addition, numerous vesicles that contain unassembled microtubules were observed within developing spermatids. Acrosome structure analysis showed normal appearance, thus excluding a crucial role of IFT81 in acrosome biogenesis. These observations showed that IFT81 is an important member of the IFT process during spermatogenesis and that its absence is associated with abnormal flagellum formation leading to male infertility. The expression levels of several IFT components in testes, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but not IFT140, were significantly reduced in homozygous mutant mice. Overall, our study demonstrates that IFT81 plays an essential role during spermatogenesis by modulating the assembly and elongation of the sperm flagella.

Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology.

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

Spermatogenesis as a tool for staging gonad development in the gonochoric appendicularian Oikopleura dioica Fol 1872.

Oikopleura dioica, the only gonochoric species among appendicularians, has a spematozoon with a mid-piece and a conspicuous acrosome that, during fertilisation, undergoes a reaction forming an acrosomal process. To provide more insight into the spermatogenesis of a holoplanktonic tunicate species that completes its life cycle in three to five days, changes in the testis during individual growth have been examined. Spermatogenesis has been subdivided into seven stages based on ultrastructural features during the formation and organisation of the male gonad and the relationships between its macroscopic anatomy and the events of sperm differentiation. Gametes undergo highly synchronised differentiation due to the presence of widespread syncytial structures. Both meiosis and spermiogenesis are brief, and the passage from spermatocytes to spermatids involves a progressive segregation of the germ cells from the syncytial mass with the formation of large cytoplasmic bridges and volume reduction for nucleus compacting and cytoplasmic material changing. The nucleus is small and penetrated anteriorly by a complex acrosome and posteriorly by the distal centriole and part of the flagellum. In spermatids, the single, large mitochondrion appears laterally to the nucleus, and finally, in spermatozoa, it migrates into the mid-piece, wrapping the proximal portion of the axoneme. Because this mitochondrial position is reached only in the late phases of spermatogenesis, it suggests that appendicularians have derived oligopyrenic sperms in which the small nucleus results from adaptation to the assembly of numerous spermatozoa inside the narrow space of the testis compacted in the genital cavity. The formulation of a staging system of gonad development in a model tunicate species known for having the most compacted genome in chordates led to a comparison of histological observations with recent molecular data, improving the characterisation of its biology and life cycle in light of evolutionary implications.

Sexual dimorphism in the width of the mouse synaptonemal complex.

Sexual dimorphism has been used to describe morphological differences between the sexes, but can be extended to any biologically related process that varies between males and females. The synaptonemal complex (SC) is a tripartite structure that connects homologous chromosomes in meiosis. Here, aided by super-resolution microscopy techniques, we show that the SC is subject to sexual dimorphism, in mouse germ cells. We have identified a significantly narrower SC in oocytes and have established that this difference does not arise from a different organization of the lateral elements nor from a different isoform of transverse filament protein SYCP1. Instead, we provide evidence for the existence of a narrower central element and a different integration site for the C-termini of SYCP1, in females. In addition to these female-specific features, we speculate that post-translation modifications affecting the SYCP1 coiled-coil region could render a more compact conformation, thus contributing to the narrower SC observed in females.

Knockdown of IP3R1 disrupts tubulobulbar complex-ectoplasmic reticulum contact sites and the morphology of apical processes encapsulating late spermatids†.

Tubulobulbar complexes (TBCs) internalize intercellular junctions during sperm release. One of the characteristic features of TBCs is that they form "bulbs" or swollen regions that have well-defined membrane contact sites (MCS) with adjacent cisternae of endoplasmic reticulum. Previously, we have localized the IP3R calcium channel to the TBC bulb-ER contacts and have hypothesized that fluctuations in local calcium levels may facilitate the maturation of TBC bulbs into putative endosomes, or alter local actin networks that cuff adjacent tubular regions of the TBCs. To test this, we injected the testes of Sprague Dawley rats with small interfering RNAs (siRNAs) against IP3R1 and processed the tissues for either western blot, immunofluorescence, or electron microscopy. When compared to control testes injected with nontargeting siRNAs, Sertoli cells in knocked-down testes showed significant morphological alterations to the actin networks including a loss of TBC actin and the appearance of ectopic para-crystalline actin bundles in Sertoli cell stalks. There also was a change in the abundance and distribution of TBC-ER contact sites and large internalized endosomes. This disruption of TBCs resulted in delay of the withdrawal of apical processes away from spermatids and in spermiation. Together, these findings are consistent with the hypothesis that calcium exchange at TBC-ER contacts is involved both in regulating actin dynamics at TBCs and in the maturing of TBC bulbs into endosomes. The results are also consistent with the hypothesis that TBCs are part of the sperm release mechanism.

Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research?

The significant rise in male infertility disorders over the years has led to extensive research efforts to recapitulate the process of male gametogenesis in vitro and to identify essential mechanisms involved in spermatogenesis, notably for clinical applications. A promising technology to bridge this research gap is organ-on-chip (OoC) technology, which has gradually transformed the research landscape in ART and offers new opportunities to develop advanced in vitro culture systems. With exquisite control on a cell or tissue microenvironment, customized organ-specific structures can be fabricated in in vitro OoC platforms, which can also simulate the effect of in vivo vascularization. Dynamic cultures using microfluidic devices enable us to create stimulatory effect and non-stimulatory culture conditions. Noteworthy is that recent studies demonstrated the potential of continuous perfusion in OoC systems using ex vivo mouse testis tissues. Here we review the existing literature and potential applications of such OoC systems for male reproduction in combination with novel bio-engineering and analytical tools. We first introduce OoC technology and highlight the opportunities offered in reproductive biology in general. In the subsequent section, we discuss the complex structural and functional organization of the testis and the role of the vasculature-associated testicular niche and fluid dynamics in modulating testis function. Next, we review significant technological breakthroughs in achieving in vitro spermatogenesis in various species and discuss the evidence from microfluidics-based testes culture studies in mouse. Lastly, we discuss a roadmap for the potential applications of the proposed testis-on-chip culture system in the field of primate male infertility, ART and reproductive toxicology.

Comparative gonadotoxicity of the chemotherapy drugs cisplatin and carboplatin on prepubertal mouse gonads.

The treatment of childhood cancer with chemotherapy drugs can result in infertility in adulthood. Newer generations of drugs are developed to replace parent drugs, with the potential benefits of less toxic side effects. For platinum alkylating-like drugs, in contrast to the parent compound cisplatin, the newer-generation drug carboplatin is reported to have reduced toxicity in some respects, despite being administered at 5-15 times higher than the cisplatin dose. Whether carboplatin is also less toxic than cisplatin to the reproductive system is unknown. Here we compare the gonadotoxic impact of cisplatin and carboplatin on female and male mouse prepubertal gonads. In vitro cultured CD1 mouse ovaries or testis fragments were exposed to either cisplatin or carboplatin for 24 h on Day 2 of culture and analysed by Day 6. A dose response for each drug was determined for the ovary (0.5, 1 & 5 µg/ml cisplatin and 1, 5 & 10 µg/ml carboplatin) and the testis (0.01, 0.05 & 0.1 µg/ml cisplatin and 0.1, 0.5 & 1 µg/ml carboplatin). For the ovary, unhealthy follicles were evident from 1 µg/ml cisplatin (73% unhealthy, P = 0.001) and 5 µg/ml carboplatin (84% unhealthy, P = 0.001), with a concomitant reduction in follicle number (P = 0.001). For the testis, the proliferating germ cell population was significantly reduced from 0.05 µg/ml cisplatin (73% reduction, P = 0.001) and 0.5 µg/ml carboplatin (75% reduction, P = 0.001), with no significant impact on the Sertoli cell population. Overall, results from this in vitro animal model study indicate that, at patient equivalent concentrations, carboplatin is no less gonadotoxic than cisplatin.