Postmeiotic Germ Cells Research Articles

Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.

Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

Testis on a chip—a microfluidic three-dimensional culture system for the development of spermatogenesis in-vitro

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

ADAD2 regulates heterochromatin in meiotic and post-meiotic male germ cells via translation of MDC1.

ABSTRACTMale germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell-specific RNA-binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mouse mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.

MALE HAPLOID CELLS THROUGH DIRECT SPHERIFICATION

We designed a 3D culture system based on direct spherification to promote neogametogenesis of mouse embryonic stem cells (mESCs) into putative post-meiotic male germ cells, confirmed by expression of acrosin, and utilize these cells to fertilize oocytes.

O-101 Neospermatogenesis benefits from a three-dimensional culture system

Abstract Study question Does a three-dimensional (3D) culture system increase the efficiency of male germline differentiation of mouse embryonic stem cells (mESC) over a bidimensional method? Summary answer Our 3D culture system based on direct spherification proves superior to the standard bidimensional plating in promoting neogametogenesis of mESC into post-meiotic male germ cells. What is known already Two-dimensional monolayer cell cultures are common in stem cell research. However, this method does not replicate a physiological 3D spatial relationship and may provide an inaccurate replication of in vivo environments. A 3D spherical structure allows us to mimic the seminiferous tubule, the site of in vivo spermatogenesis. By using spheroids as a scaffold to replicate cell culture systems, we can study spermatogenesis in a controlled setting. Direct spherification, a technique commonly used in molecular gastronomy, provides an opportunity to create spheroids that mimic in vivo events that materialize in the lab Study design, size, duration mESCs were initially cultured on a 6-well plate coated with fibroblasts and inserted into sodium alginate spheres. To coax differentiation, spheres (3 to 6 mm in diameter) were plunged directly into differentiation medium (DM) while the control mESC in 6-well dishes were layered with it. Cells obtained from both culture systems were tested by biomarkers for different germ cell stages Participants/materials, setting, methods Bidimensional mESC at 80% confluence were differentiated either on a plate or spherified for a 3D culture. Both systems underwent the same timeline of exposure to EpiLC medium with Activin A, bFGF and KSR for 3 days and PGCLC medium with BMP4, LIF, SCF and EGF for 7 days. Differentiated cells were retrieved from each method at day 3 and day 10 to assess for germ line differentiation markers, DAZL, VASA and BOULE Main results and the role of chance Under optic visualization through the sphere wall, cellular aggregation was seen on day 2 of culturing in EpiLC medium while this phenomenon was not observed on bidimensional plating. In the conventional method, cells expressed 7% DAZL (spermatogonium cell stage) and 1% VASA (pre-spermatid cell stage) whereas in direct spherification, cells expressed 20% DAZL (P < 0.001) and 15% VASA positivity (P < 0.0001). To further compare the different methods in later stages of germ-line differentiation, the remaining spheres were cultured in PGCLC medium for 7 days. At day 10, isolated cells were assessed for VASA and DAZL again. In the conventional method, 23% of cells expressed positivity for VASA and 29% DAZL whereas direct spherification achieved a positivity rate of 43% for VASA (P < 0.005) and 45% for DAZL (P < 0.005). This increased expression in both VASA and DAZL signify the increased number of cells undergoing germline differentiation. Additionally, BOULE was assessed for the presence of meiotic cells such as the spermatocyte. The conventional method yielded < 1% BOULE positivity whereas in direct spherification, there was 10% positivity (P < 0.005). Direct spherifcation result shows that differentiation almost doubled in comparison to the conventional method, yielding more post-meiotic cells in the same amount of time Limitations, reasons for caution Despite a higher differentiation rate in direct spherification, these cells would still need to be tested for their fertilization potential. The ability to achieve fertilization, blastocysts and live pups would provide final proof and reliability of this method of neogametogenesis Wider implications of the findings Differentiating ESCs through direct spherification provides an alternative to studying intercellular relationships. This provides an opportunity to study spermatogenesis in more detail by replicating the microenvironment of the seminiferous tubule. Once embryo developmental competence of the de novo gamete is confirmed, this may open a new chapter in human reproduction Trial registration number N/A

Germ Cell Derivation from Pluripotent Stem Cells for Understanding In Vitro Gametogenesis.

Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells. Here, we review the recent advances in in vitro male and female germ cell derivation from PSCs and discuss how this technique is used to understand the biological mechanism of gamete development and gain insight into its application in infertility.

Peptidyl Arginine Deiminase 2 (PADI2) is Expressed in Post-Meiotic Germ Cells in the Mouse Testis and is Localized Heavily on the Acrosomal Region of Spermatozoa

Peptidyl Arginine Deiminase 2 (PADI2) is a widely expressed Ca 2+ ion-dependent enzyme in rodents and humans belonging to Peptidyl Arginine Deiminases (PAD) family. It regulates various cellular processes like proliferation, differentiation, apoptosis, migration, epithelial-mesenchymal transition and chromatin organization. Altered expression of PADI2 was associated with various autoimmune diseases, neurological disorders and different types of cancers. Based on our previously published miRNA-mRNA network during the first wave of spermatogenesis in the mouse, Padi2 appeared to be a common potential target of miR-34c and miR-449a in the mouse testes. In the present study, the expression of Padi2 in the mouse testes during the first wave of spermatogenesis was evaluated using real-time PCR, western blot analysis and immunohistochemistry. Transcript levels of Padi2 showed progressive down-regulation during the first wave of spermatogenesis. However, we detected a progressive increase in the levels of PADI2 as the first wave of spermatogenesis progressed, with heavy expression of this protein in post-meiotic germ cells. Additionally, a prominent localization of PADI2 in the acrosomal region of late spermatids and spermatozoa was identified through immunohistochemical analysis. Padi2 expression was identified in germ cell-derived cell lines, C18-4 and GC-1 spg as well. Thus, the present study illustrates for the first time the expression of PADI2 in germ cells in the testis and its predominant localization on the acrosome region of spermatozoa, suggesting its potential role in fertilization.

Identification of testicular Foxq1 as a critical modulator of lactate metabolism in mouse Sertoli cells.

Postmeiotic germ cells require the lactate produced by the adjacent Sertoli cells (SCs) as their sole energy fuels. Lactate production in SCs is elaborately regulated by monitoring the transcription of the lactate dehydrogenase A (Ldha) gene. However, the transcription factors that are responsible for the control of Ldha transcription in SCs remain ill defined. Herein, the expression of forkhead box Q1 (FOXQ1), a central modulator of glucose metabolism in liver, was demonstrated in mouse testis throughout postnatal development, with maximum levels in adult specimens. At this age, FOXQ1 was immunolocalized in the nuclei of the functionally mature SCs. Testicular levels of FOXQ1 were overtly modulated by germ cells (GCs)-derived IL-1α, in a dose- and time-dependent manner. To further clarify the biological functions of FOXQ1, we disrupted the mouse Foxq1 gene using a Cas9/RNA-mediated gene targeting strategy. Foxq1-/- males were subfertile and showed oligoasthenozoospermia due to lactate deficiency. Moreover, we provided the molecular evidence that FOXQ1 may regulate lactate production by directly targeting the transactivation of the Ldha gene in SCs. From a functional standpoint, overexpression of the exogenous Ldha ameliorated Foxq1 deficiency-impaired lactate synthesis in the SCsFoxq1-/- cells. Thus, these findings collectively underscore a reproductive facet of this recently characterized transcription factor, which may operate as a novel transcriptional integrator linking energy homeostasis and nursery function in SCs.

Integrating miRNA and mRNA Profiling to Assess the Potential miRNA-mRNA Modules Linked With Testicular Immune Homeostasis in Sheep.

Beyond its well-known role in spermatogenesis and androgen production, mammalian testes are increasingly recognized as an immune-privileged organ for protecting autoantigenic germ cells, especially meiotic and postmeiotic germ cells, from systemic immune responses. Despite its importance, the molecular mechanisms underlying this regulation in mammals, including sheep, are far from known. In this study, we searched for the genes associated with testicular immune privilege and assessed their possible modulating mechanisms by analyzing systematic profiling of mRNAs and miRNAs on testicular tissues derived from prepubertal and postpubertal Tibetan sheep acquired by RNA sequencing. We identified 1,118 differentially expressed (DE) mRNAs associated with immunity (245 increased mRNAs and 873 decreased mRNAs) and 715 DE miRNAs (561 increased miRNAs and 154 decreased miRNAs) in postpubertal testes compared with prepuberty. qPCR validations for 20 DE mRNAs and 16 miRNAs showed that the RNA-seq results are reliable. By using Western blot, the postpubertal testes exhibited decreased protein abundance of CD19 and TGFBR2 (two proteins encoded by DE mRNAs) when compared with prepuberty, consistent with mRNA levels. The subsequent immunofluorescent staining showed that the positive signals for the CD19 protein were observed mainly in Sertoli cells and the basement membrane of pre- and postpubertal testes, as well as the prepubertal testicular vascular endothelium. The TGFBR2 protein was found mostly in interstitial cells and germ cells of pre- and postpubertal testes. Functional enrichment analysis indicated that DE mRNAs were mainly enriched in biological processes or pathways strongly associated with the blood–testis barrier (BTB) function. Many decreased mRNAs with low expression abundance were significantly enriched in pathways related to immune response. Also, multiple key miRNA-target negative correlation regulatory networks were subsequently established. Furthermore, we verified the target associations between either oar-miR-29b or oar-miR-1185-3p and ITGB1 by dual-luciferase reporter assay. Finally, a putative schematic model of the miRNA-mRNA-pathway network mediated by immune homeostasis-related genes was proposed to show their potential regulatory roles in sheep testicular privilege. Taken together, we conclude that many immune-related genes identified in this study are negatively regulated by potential miRNAs to participate in the homeostatic regulation of testicular immune privilege of sheep by sustaining BTB function and inhibiting immune responses under normal physiological conditions. This work offers the first global view of the expression profiles of miRNAs/mRNAs involved in sheep testicular immune privilege and how the genes potentially contribute to immune-homeostatic maintenance.

Effect of Cryptorchidism on the Histomorphometry, Proliferation, Apoptosis, and Autophagy in Boar Testes.

Simple SummaryBody temperature has detrimental effects on sperm quality in mammalian species, including pigs. However, the molecular mechanism of this is not yet well understood. Cryptorchidism is when one or both testes fail to descend into the scrotum, which leads the testes to be exposed to the body temperature. The aims of present study were to investigate the effect of body temperature on the histomorphometry, apoptosis and the expression of the proliferation-associated protein PCNA and the autophagy-associated protein LC3 in spontaneous unilateral cryptorchid boar testes. Our findings showed that cryptorchidism had no evident influence on the number of Sertoli cells in boars. In cryptorchid testes, spermatogonia markedly decreased and the seminiferous tubule contained only a few spermatogonia, but did not contain post-meiotic germ cells. The altered seminiferous epithelium of the cryptorchid testis showed a low proliferation of its spermatogonia, with apoptosis and autophagy like that of scrotal testis, which probably entailed a gradual degeneration of the epithelium and the impossibility of its recovery. Although the number of Sertoli cells did not change, it was likely that their functionality was altered and that this affected the proliferation capacity of spermatogonia, causing the arrest of spermatogenesis.Spontaneous unilateral cryptorchid boars have one testis in the abdomen or inguinal canal, causing its temperature to be at or near the body temperature, which impairs spermatogenesis, although the histomorphometry and molecular mechanisms underlying this process remain unclear. The aim of the present study was to determine the histomorphometry, proliferation, apoptosis, and autophagy alterations in spermatogonia and Sertoli cells in unilateral cryptorchid, scrotal (contrascrotal), and preweaning piglet (preweaning) testes. Histomorphometrical analysis of cryptorchid testes showed that the seminiferous tubules contained only Sertoli cells and a few spermatogonia, but did not contain post-meiotic germ cells. The number of spermatogonia markedly decreased, and the number of Sertoli cells did not change remarkably in cryptorchid testes. TUNEL assay results showed that apoptosis signals were predominantly observed in spermatogonia. In cryptorchid and contrascrotal testes, proliferating cell nuclear antigen (PCNA) and LC3 were located in spermatogonia. The number of PCNA-positive, TUNEL-positive, and LC3-positive germ cells was low, and the protein and mRNA levels of PCNA and LC3 were significantly decreased in cryptorchid testes. Taken together, the number of Sertoli cells did not change remarkably, whereas the number of germ cells decreased in the cryptorchid testes, compared with that in the contrascrotal testes. Insufficient proliferation, excessive apoptosis, and autophagy were involved in the regulation of the decrease in spermatogonia in cryptorchid boar testes.

HDAC3 controls male fertility through enzyme-independent transcriptional regulation at the meiotic exit of spermatogenesis.

The transition from meiotic spermatocytes to postmeiotic haploid germ cells constitutes an essential step in spermatogenesis. The epigenomic regulatory mechanisms underlying this transition remain unclear. Here, we find a prominent transcriptomic switch from the late spermatocytes to the early round spermatids during the meiotic-to-postmeiotic transition, which is associated with robust histone acetylation changes across the genome. Among histone deacetylases (HDACs) and acetyltransferases, we find that HDAC3 is selectively expressed in the late meiotic and early haploid stages. Three independent mouse lines with the testis-specific knockout of HDAC3 show infertility and defects in meiotic exit with an arrest at the late stage of meiosis or early stage of round spermatids. Stage-specific RNA-seq and histone acetylation ChIP-seq analyses reveal that HDAC3 represses meiotic/spermatogonial genes and activates postmeiotic haploid gene programs during meiotic exit, with associated histone acetylation alterations. Unexpectedly, abolishing HDAC3 catalytic activity by missense mutations in the nuclear receptor corepressor (NCOR or SMRT) does not cause infertility, despite causing histone hyperacetylation as HDAC3 knockout, demonstrating that HDAC3 enzyme activity is not required for spermatogenesis. Motif analysis of the HDAC3 cistrome in the testes identified SOX30, which has a similar spatiotemporal expression pattern as HDAC3 during spermatogenesis. Depletion of SOX30 in the testes abolishes the genomic recruitment of the HDAC3 to the binding sites. Collectively, these results establish the SOX30/HDAC3 signaling as a key regulator of the transcriptional program in a deacetylase-independent manner during the meiotic-to-postmeiotic transition in spermatogenesis.

Of rodents and ruminants: a comparison of small noncoding RNA requirements in mouse and bovine reproduction.

Ruminants are major producers of meat and milk, thus managing their reproductive potential is a key element in cost-effective, safe, and efficient food production. Of particular concern, defects in male germ cells and female germ cells may lead to significantly reduced live births relative to fertilization. However, the underlying molecular drivers of these defects are unclear. Small noncoding RNAs, such as piRNAs and miRNAs, are known to be important regulators of germ-cell physiology in mouse (the best-studied mammalian model organism) and emerging evidence suggests that this is also the case in a range of ruminant species, in particular bovine. Similarities exist between mouse and bovids, especially in the case of meiotic and postmeiotic male germ cells. However, fundamental differences in small RNA abundance and metabolism between these species have been observed in the female germ cell, differences that likely have profound impacts on their physiology. Further, parentally derived small noncoding RNAs are known to influence early embryos and significant species-specific differences in germ-cell born small noncoding RNAs have been observed. These findings demonstrate the mouse to be an imperfect model for understanding germ-cell small noncoding RNA biology in ruminants and highlight the need to increase research efforts in this underappreciated aspect of animal reproduction.

Biparental contributions of the H2A.B histone variant control embryonic development in mice.

Histone variants expand chromatin functions in eukaryote genomes. H2A.B genes are testis-expressed short histone H2A variants that arose in placental mammals. Their biological functions remain largely unknown. To investigate their function, we generated a knockout (KO) model that disrupts all 3 H2A.B genes in mice. We show that H2A.B KO males have globally altered chromatin structure in postmeiotic germ cells. Yet, they do not show impaired spermatogenesis or testis function. Instead, we find that H2A.B plays a crucial role postfertilization. Crosses between H2A.B KO males and females yield embryos with lower viability and reduced size. Using a series of genetic crosses that separate parental and zygotic contributions, we show that the H2A.B status of both the father and mother, but not of the zygote, affects embryonic viability and growth during gestation. We conclude that H2A.B is a novel parental-effect gene, establishing a role for short H2A histone variants in mammalian development. We posit that parental antagonism over embryonic growth drove the origin and ongoing diversification of short histone H2A variants in placental mammals.

Impact of Temperature and Time Interval Prior to Immature Testicular-Tissue Organotypic Culture on Cellular Niche

Cryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis.

Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post-meiotic progression was impaired. As potential key inducers of the mitosis-meiosis switch, type 2 cannabinoid receptor (CB2 ) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB2 activation on the differentiation of spermatogonia until elongated spermatids. A first set of cultured testicular explants of 6.5days post-partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10nm, 100nm, 1µm, 10µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp). CB2 was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB2 by 1μm of the agonist JWH133, percentage of zygotene spermatocyte I increased. After 30days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post-meiotic germ cells were observed. We showed that the activation of CB2 improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.

Distinct expression and localization patterns of HSP70 in developmental reproductive organs of rams.

Heat shock protein 70 (HSP70) has been widely reported to play a vital role in maintaining intracellular homeostasis, mainly through cellular protection and immune regulation. The expression and function of HSP70 can vary depending upon species and age. To explore the expression signatures and regulatory functions of HSP70 in the reproductive organs of male sheep, we evaluated the expression and distribution patterns of HSP70 in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at three developmental stages (i.e., 3months, 1year and 3years after birth) by qRT-PCR, Western blot and immunofluorescence. HSP70 was found to be expressed in testes, caput, corpus, and cauda epididymides throughout the developmental stages but is mainly expressed postpuberty (1year and 3years old). Immunofluorescence results revealed that in the testes, a positive reaction for HSP70 protein was mainly seen in round spermatids and luminal sperms from the groups aged 1year and 3years. In caput epididymides, the positive signals for HSP70 protein was notably observed in sperm and principal cells of the epididymal epithelium from the groups aged 1year and 3years, and positive signals in the epididymal interstitium were found in all three age groups. In corpus and cauda epididymides, HSP70 protein was present in the epididymal epithelium and interstitium, and the positive signals gradually increased with age. In conclusion, these findings suggest that Tibetan sheep HSP70 may play a crucial role in further development and maturation of postmeiotic germ cells and participate in regulation of intraepididymal homeostasis maintenance in Tibetan sheep.

ADAD1 and ADAD2, testis-specific adenosine deaminase domain-containing proteins, are required for male fertility

Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.

Nigral and ventral tegmental area lesioning induces testicular and sperm morphological abnormalities in a rotenone model of Parkinson’s disease

Although sexual health is affected by Parkinson’s disease (PD), the effect on testicular health and/or sperm quality is not well discussed. After 21 days of rotenone lesioning, we observed dopaminergic neuronal degeneration in the substantia nigra and hypothalamus. There were minimal SPACA-1-expressing epididymal spermatozoa with morphological abnormalities, scanty luminal spermatozoa and reduced testicular spermatids and post-meiotic germ cells indicating hypospermatogenesis. Occludin-expressing sertoli cells were dispersed over a wide area indicating compromised blood-testes barrier. Activated caspase-3 expression was intense while immunoreactivity of spermatogenic-enhancing SRY and GADD45 g was weak. Although serum follicle stimulating hormone level was not affected, the lesion was associated with reduced serum testosterone level, testicular oxidative damage and inhibition of acetylcholinesterase activity, even when rotenone was not detected in the testes. Together, dopaminergic lesions may mediate testicular and sperm abnormalities via the brain-hypothalamic-testicular circuit independent of the pituitary, thereby establishing a causal link between Parkinsonism and reproductive dysfunction.

Deletion of inositol polyphosphate 4-phosphatase type-II B affects spermatogenesis in mice.

Inositol polyphosphate-4-phosphatase type II (INPP4B) is a dual-specificity phosphatase that acts as a tumor suppressor in multiple cancers. INPP4B dephosphorylates phospholipids at the 4th position of the inositol ring and inhibits AKT and PKC signaling by hydrolyzing of PI(3,4)P2 and PI(4,5)P2, respectively. INPP4B protein phosphatase targets include phospho-tyrosines on Akt and phospho-serine and phospho-threonine on PTEN. INPP4B is highly expressed in testes, suggesting its role in testes development and physiology. The objective of this study was to determine whether Inpp4b deletion impacts testicular function in mice. In testis, Inpp4b expression was the highest in postmeiotic germ cells in both mice and men. The testes of Inpp4b knockout male mice were significantly smaller compared to the testes of wild-type (WT) males. Inpp4b-/- males produced fewer mature sperm cells compared to WT, and this difference increased with age and high fat diet (HFD). Reduction in early steroidogenic enzymes and luteinizing hormone (LH) receptor gene expression was detected, although androgen receptor (AR) protein level was similar in WT and Inpp4b-/- testes. Germ cell apoptosis was significantly increased in the knockout mice, while expression of meiotic marker γH2A.X was decreased. Our data demonstrate that INPP4B plays a role in maintenance of male germ cell differentiation and protects testis functions against deleterious effects of aging and high fat diet.

ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.