Mammalian Testis Research Articles

The Seminiferous Epithelial Cycle of Spermatogenesis: Role of Non-receptor Tyrosine Kinases.

Non-receptor tyrosine kinases (NRTKs) are implicated in various biological processes including cell proliferation, differentiation, survival, and apoptosis, as well as cell adhesion and movement. NRTKs are expressed in all mammals and in different cell types, with extraordinarily high expression in the testis. Their association with the plasma membrane and dynamic subcellular localization are crucial parameters in their activation and function. Many NRTKs are found in endosomal protein trafficking pathways, which suggests a novel mechanism to regulate the timely junction restructuring in the mammalian testis to facilitate spermiation and germ cell transport across the seminiferous epithelium.

Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells.

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3β-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.

T Lymphocytes and Testicular Immunity: A New Insight into Immune Regulation in Testes.

The immune privilege of the testes is necessary to prevent immune attacks to gamete-specific antigens and paternal major histocompatibility complex (MHC) antigens, allowing for normal spermatogenesis. However, infection and inflammation of the male genital tract can break the immune tolerance and represent a significant cause of male infertility. Different T cell subsets have been identified in mammalian testes, which may be involved in the maintenance of immune tolerance and pathogenic immune responses in testicular infection and inflammation. We reviewed the evidence in the published literature on different T subtypes (regulatory T cells, helper T cells, cytotoxic T cells, γδ T cells, and natural killer T cells) in human and animal testes that support their regulatory roles in infertility and the orchitis pathology. While many in vitro studies have indicated the regulation potential of functional T cell subsets and their possible interaction with Sertoli cells, Leydig cells, and spermatogenesis, both under physiological and pathological processes, there have been no in situ studies to date. Nevertheless, the normal distribution and function of T cell subsets are essential for the immune privilege of the testes and intact spermatogenesis, and T cell-mediated immune response drives testicular inflammation. The distinct function of different T cell subsets in testicular homeostasis and the orchitis pathology suggests a considerable potential of targeting specific T cell subsets for therapies targeting chronic orchitis and immune infertility.

Microtubule-associated proteins (MAPs) in microtubule cytoskeletal dynamics and spermatogenesis.

The microtubule (MT) cytoskeleton in Sertoli cells, a crucial cellular structure in the seminiferous epithelium of adult mammalian testes that supports spermatogenesis, was studied morphologically decades ago. However, its biology, in particular the involving regulatory biomolecules and the underlying mechanism(s) in modulating MT dynamics, are only beginning to be revealed in recent years. This lack of studies in delineating the biology of MT cytoskeletal dynamics undermines other studies in the field, in particular the plausible therapeutic treatment and management of male infertility and fertility since studies have shown that the MT cytoskeleton is one of the prime targets of toxicants. Interestingly, much of the information regarding the function of actin-, MT- and intermediate filament-based cytoskeletons come from studies using toxicant models including some genetic models. During the past several years, there have been some advances in studying the biology of MT cytoskeleton in the testis, and many of these studies were based on the use of pharmaceutical/toxicant models. In this review, we summarize the results of these findings, illustrating the importance of toxicant/pharmaceutical models in unravelling the biology of MT dynamics, in particular the role of microtubule-associated proteins (MAPs), a family of regulatory proteins that modulate MT dynamics but also actin- and intermediate filament-based cytoskeletons. We also provide a timely hypothetical model which can serve as a guide to design functional experiments to study how the MT cytoskeleton is regulated during spermatogenesis through the use of toxicants and/or pharmaceutical agents.

Effect of feeding linseed diet on testis development, antioxidant capacity, and epididymal cauda sperm concentration in Chinese Hu lamb

Polyunsaturated fatty acids (PUFAs) are essential for mammalian testis development and sperm function. However, PUFAs that are contained in linseed oil are easily oxidized in the diet and biohydrogenated in the rumen. In this study, we investigated the effect of linseed as a source of PUFAs on the antioxidant capacity and testis development in Hu lamb. Seventy-five 3-month-old lambs were randomly assigned to three groups. Within each treatment group, 25 lambs were allocated to five pens (five lambs per pen). The lambs in the control group were fed a control diet without linseed for 42 days from D22 to D63. Group I (BS28) was fed a control diet from D22 to D35 and 8% linseed diet from D36 to D63. Group II (BS42) was fed an 8% linseed diet for 42 days from D22 to D63. After 63-day feeding trial, all lambs except the heaviest and lightest in each pen were humanely slaughtered and investigated. Results revealed that feeding linseed did not affect the body weight, scrotal circumference, and testis weight, whereas feeding linseed for 42 days increased the epididymis weight (37.85 ± 1.61 g vs. 32.09 ± 1.06 g, P < 0.05) compared with the control group. Feeding lambs with linseed for 42 days also significantly upregulated the expression of antioxidative (glutathione peroxidase 4 and copper-zinc superoxide dismutase), steroidogenesis (3β-hydroxysteroid dehydrogenase and steroid acute regulatory protein), and PUFA metabolism-related genes (fatty acid desaturase 2 and elongation of very long-chain fatty acid protein 2) and proliferating cell nuclear antigen mRNA (P < 0.05). It also increased the relative expression of mitochondrial DNA (P < 0.05), total antioxidant capacity (0.230 ± 0.019 mmol/mgprot vs. 0.175 ± 0.011 mmol/mgprot, P < 0.05), and superoxide dismutase (1661.467 ± 147.117 U/mgprot vs. 1158.891 ± 98.850 U/mgprot, P < 0.05) in testicular tissue but decreased the cholesterol concentration (0.331 ± 0.073 mmol/mgprot vs. 0.671 ± 0.092 mmol/mgprot, P < 0.05) compared with the control group. Therefore, feeding lambs with linseed for 42 days stimulated seminiferous tubule development and increased the number of Sertoli cells (20.71 ± 0.89 vs. 17.6 ± 0.73, P < 0.05), epididymal cauda lumina diameter (638.26 ± 22.32 μm vs. 444.41 ± 34.80 μm, P < 0.05), and the number of sperm in the epididymal cauda (68.91 ± 7.06 × 108/g vs. 36.61 ± 7.50 × 108/g). All these results suggested that feeding linseed in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and PUFA metabolism-related genes; increased the antioxidant capacity in lamb’s testis; and contributed to testis development and spermatogenesis.

Histological Analysis, Bioinformatics Profile, and Expression of Methylenetetrahydrofolate Reductase (MTHFR) in Bovine Testes

Simple SummaryTo date, several genes have been sequenced but their corresponding protein characteristics remain unknown. This study highlights the histological structure of bovine (yellow-cattle and yak) testes as a build-up to exploring the bioinformatics profile and expression characteristics of methylenetetrahydrofolate reductase (MTHFR) in bovine testes. Our findings suggest that yellow-cattle testis have similar anatomical characteristics with that of yak, except for the weight or size, for which that of yellow-cattle is significantly higher or greater than yak. We also found that the secondary and 3D protein structures of MTHFR were similar to that of humans, with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. Moreover, MTHFR mRNA expression was higher in adult yellow-cattle and yak compared to their juvenile ones, however, its protein expression was higher but not statistically significant in adult yellow-cattle and yak compared to the juvenile ones. This provides a basis for further investigations into the regulatory function of MTHFR in bovine testes.Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes, exerts a direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤2 years juvenile (y-c, n = 3; yak, n = 3) and ≥3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher, but not statistically significant, in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis.

SummarySpermatogonial stem cells (SSCs) both self-renew and give rise to progenitors that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable with that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly for H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs or lose regenerative capacity and initiate lineage commitment as progenitors.

E4 Transcription Factor 1 (E4F1) Regulates Sertoli Cell Proliferation and Fertility in Mice.

Simple SummaryMale fertility relies on the generation of functional sperm in seminiferous tubules of the testis. In mammals, Sertoli cells are the only somatic cells that directly interact with spermatogenic cells. Compelling evidences suggest that the number of Sertoli cells determines testis size and sperm output, however, molecular mechanisms regulating Sertoli cell proliferation and maturation are not well-understood. Using a Sertoli cell specific loss-of-function approach, here we showed that transcription factor E4F1 played an important role in murine Sertoli cell proliferation. Compared with their littermate control, E4f1 conditional knockout male mice sired a significantly low number of pups. E4f1 deletion resulted in reduced Sertoli cell number and testis size. Further analyses revealed that E4f1 deletion affected Sertoli cell proliferation in the neonatal testis and caused an increase in apoptosis of spermatogenic cells without affecting normal development of spermatogonia, meiotic and post-meiotic germ cells. These findings have shed new light on molecular controlling of spermatogenesis in mice and a similar mechanism likely exists in other animals.In the mammalian testes, Sertoli cells are the only somatic cells in the seminiferous tubules that provide structural, nutritional and regulatory support for developing spermatogenic cells. Sertoli cells only proliferate during the fetal and neonatal periods and enter a quiescent state after puberty. Functional evidences suggest that the size of Sertoli cell population determines sperm production and fertility. However, factors that direct Sertoli cell proliferation and maturation are not fully understood. Transcription factor E4F1 is a multifunctional protein that serves essential roles in cell fate decisions and because it interacts with pRB, a master regulator of Sertoli cell function, we hypothesized that E4F1 may have a functional role in Sertoli cells. E4f1 mRNA was present in murine testis and immunohistochemical staining confirmed that E4F1 was enriched in mature Sertoli cells. We generated a conditional knockout mouse model using Amh-cre and E4f1flox/flox lines to study E4F1 fucntion in Sertoli cells and the results showed that E4f1 deletion caused a significant reduction in testis size and fertility. Further analyses revealed that meiosis progression and spermiogenesis were normal, however, Sertoli cell proliferation was impaired and germ cell apoptosis was elevated in the testis of E4f1 conditional knockout mice. On the basis of these findings, we concluded that E4F1 was expressed in murine Sertoli cells and served important functions in regulating Sertoli cell proliferation and fertility.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood–testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell–cell and also Sertoli–spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Early vertebrate origin of CTCFL, a CTCF paralog, revealed by proximity-guided shark genome scaffolding

The nuclear protein CCCTC-binding factor (CTCF) contributes as an insulator to chromatin organization in diverse animals. The gene encoding this protein has a paralog which was first identified to be expressed exclusively in the testis in mammals and designated as CTCFL (also called BORIS). CTCFL orthologs were reported only among amniotes, and thus CTCFL was once thought to have arisen in the amniote lineage. In this study, we identified elasmobranch CTCFL orthologs, and investigated its origin with the aid of a shark genome assembly improved by proximity-guided scaffolding. Our analysis employing evolutionary interpretation of syntenic gene location suggested an earlier timing of the gene duplication between CTCF and CTCFL than previously thought, that is, around the common ancestor of extant vertebrates. Also, our transcriptomic sequencing revealed a biased expression of the catshark CTCFL in the testis, suggesting the origin of the tissue-specific localization in mammals more than 400 million years ago. To understand the historical process of the functional consolidation of the long-standing chromatin regulator CTCF, its additional paralogs remaining in some of the descendant lineages for spatially restricted transcript distribution should be taken into consideration.

Expression profiling of WD40 family genes including DDB1- and CUL4- associated factor (DCAF) genes in mice and human suggests important regulatory roles in testicular development and spermatogenesis

BackgroundThe WD40-repeat containing proteins, including DDB1–CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development.ResultsPublically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60–73% of the genes were highly or specifically expressed in testis. Similarly, around 74–83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes.ConclusionsWe have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

Microtubule Cytoskeleton and Spermatogenesis-Lesson From Studies of Toxicant Models.

Studies have shown that mammalian testes, in particular the Sertoli cells, are highly susceptible to exposure of environmental toxicants, such as cadmium, perfluorooctanesulfonate, phthalates, 2,5-hexanedione and bisphenol A. However, important studies conducted by reproductive toxicologists and/or biologists in the past have been treated as toxicology reports per se. Yet, many of these studies provided important mechanistic insights on the toxicant-induced testis injury and reproductive dysfunction, relevant to the biology of the testis and spermatogenesis. Furthermore, recent studies have shown that findings obtained from toxicant models are exceedingly helpful tools to unravel the biology of testis function in particular spermatogenesis, including specific cellular events associated with spermatid transport to support spermiogenesis and spermiation. In this review, we critically evaluate some recent data, focusing primarily on the molecular structure and role of microtubules in cellular function, illustrating the importance of toxicant models to unravel the biology of microtubule cytoskeleton in supporting spermatogenesis, well beyond information on toxicology. These findings have opened up some potential areas of research which should be carefully evaluated in the years to come.

Higher Germline Mutagenesis of Genes with Stronger Testis Expressions Refutes the Transcriptional Scanning Hypothesis.

Why are more genes expressed in the testis than in any other organ in mammals? The recently proposed transcriptional scanning hypothesis posits that transcription alleviates mutagenesis through transcription-coupled repair so has been selected in the testis to modulate the germline mutation rate in a gene-specific manner. Here, we show that this hypothesis is theoretically untenable because the selection would be too weak to have an effect in mammals. Furthermore, the analysis purported to support the hypothesis did not control known confounding factors and inappropriately excluded genes with no observed de novo mutations. After remedying these problems, we find the human germline mutation rate of a gene to rise with its testis expression level. This trend also exists for inferred coding strand-originated mutations, suggesting that it arises from transcription-associated mutagenesis. Furthermore, the testis expression level of a gene robustly correlates with its overall expression in other organs, nullifying the need to explain the testis silencing of a minority of genes by adaptive germline mutagenesis. Taken together, our results demonstrate that human testis transcription increases the germline mutation rate, rejecting the transcriptional scanning hypothesis of extensive gene expressions in the mammalian testis.

Heat-induced changes in the expression and localisation of PGC-1α in the mice testis.

The functions of mammalian testis are temperature-sensitive. There are various testicular factors, which express in response to heat as a mechanism of defence. PGC-1α and HSP70 have poetical role in the protection from oxidative stress in various tissues, including testis. The expression of PGC-1α and HSP70 has been shown in the testis, and it has also been documented that heat modulates the expression of PGC-1α and HSP70. However, heat-dependent changes in the localisation and expression of PGC-1α have not been investigated so far. Thus, we studied the expression and localisation pattern of PGC-1α in the testis of heat-treated mice along with marker of proliferation (PCNA, GCNA), serum testosterone levels, MDA levels and HSP70. The results showed a significant increase in PGC-1α and HSP70 and MDA levels in the testis of heat-treated mice along with a decrease in PCNA, GCNA and serum testosterone levels. The immunolocalisation study showed intense immunostaining of PGC-1α in the Leydig cell and germ cells of the heat-treated testis, with pronounced damaged in the histoarchitecture. The results showed that increase expression of PGC-1α in germ cells and Leydig cells of testis could be a counter mechanism to cope up with oxidative stress in coordination with HSP70.

Ubiquitome analysis reveals the involvement of lysine ubiquitination in the spermatogenesis process of adult buffalo (Bubalus bubalis) testis.

Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In the present study, we first identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatics analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Besides, 44 ubiquitinated proteins may involve in spermatogenesis according to the SpermatogenesisOnline database, of which, the ubiquitination of HSPA2 and UCHL1 were confirmed by Immunoprecipitation (IP)/Western blot analysis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role especially spermatogenesis of lysine ubiquitination in testis in mammals.

Actin binding proteins, actin cytoskeleton and spermatogenesis – Lesson from toxicant models

Actin cytoskeleton is crucial to support spermatogenesis in the mammalian testis. However, the molecular mechanism(s) underlying changes of actin cytoskeletal organization in response to cellular events that take place across the seminiferous epithelium (e.g., self-renewal of spermatogonial stem cells, germ cell differentiation, meosis, spermiogenesis, spermiation) at specific stages of the epithelial cycle of spermatogenesis remain largely unexplored. This, at least in part, is due to the lack of suitable study models to identify the crucial regulatory proteins and to investigate how these proteins work in concert to support actin dynamics. Much of the information on the role of actin binding proteins in the literature, namely the actin bundling proteins, actin nucleation proteins and motor proteins, are either findings based on genetic models or morphological analyses. While this information is helpful to delineate the function of these proteins to support spermatogenesis, they are not helpful to identify the regulatory signaling proteins, the signaling pathways and the cascade of events to modulate actin cytoskeleton dynamics. Recent studies based on the use of toxicant models, both in vitro and in vivo, however, have bridged this gap by identifying putative regulatory and signaling proteins of actin cytoskeleton. Herein, we summarize and critically evaluate these findings. We also provide a hypothetical model by which actin cytoskeletal dynamics in Sertoli cells are regulated, which in turn supports spermatid transport across the seminiferous epithelium, and at the blood-testis barrier (BTB) during the epithelial cycle of spermatogenesis.

Endogenously produced LG3/4/5-peptide protects testes against toxicant-induced injury

Laminin-α2 chain is one of the major constituent proteins of the basement membrane in the mammalian testis. The laminin-type globular (LG) domains of LG3, 4 and 5 (LG3/4/5, an 80 kDa fragment) can be cleaved from laminin-α2 chain at the C-terminus via the action of matrix metalloproteinase 9 (MMP-9). This LG3/4/5 is a biologically active fragment, capable of modulating the Sertoli cell blood–testis barrier (BTB) function by tightening the barrier both in vitro and in vivo. Overexpression of LG3/4/5 cloned into a mammalian expression vector pCI-neo in Sertoli cells in a Sertoli cell in vitro model with a functional BTB also protected Sertoli cells from cadmium chloride (CdCl2, an environmental toxicant) mediated cell injury. Importantly, overexpression of LG3/4/5 in the testis in vivo was found to block or rescue cadmium-induced BTB disruption and testis injury. LG3/4/5 was found to exert its BTB and spermatogenesis promoting effects through corrective spatiotemporal expression of actin- and MT-based regulatory proteins by maintaining the cytoskeletons in the testis, illustrating the therapeutic implication of this novel bioactive fragment.

In‐Vivo Knockdown of Cortactin Disrupts Basal Tubulobulbar Complexes and Results in the Presence of Redundant Junction Related Ectoplasmic Specializations

In the mammalian testis, spermatogenic cells begin development in the germ cell niche or basal compartment of the seminiferous epithelium. They then translocate through massive junction complexes between neighboring Sertoli cells to enter the adluminal compartment and further differentiate. Removal of the junctions above and formation of new junctions below is necessary for the translocation of cells from one compartment to the other. Tubulobulbar complexes’ (TBCs) are Sertoli cell specific structures that are proposed to internalize intercellular junctions in the seminiferous epithelium. TBCs are actin cuffed endocytic structures that share many features in common with clathrin mediated endocytosis (CME). Both TBCs and CME vesicles begin with the association of a clathrin coated pit on the plasma membrane and the development of dendritic actin ‘cuffs’ on their neck regions. Rather than vesiculating, the necks of TBCs continue to lengthen into microns long structures, eventually developing an actin free ‘bulb’ region, which develops an ORP9 positive membrane contact site with an associated leaflet of endoplasmic reticulum. One of the proteins known to be highly specific for the dendritic actin around TBCs is the actin related protein, cortactin. It previously has been shown that knockdown of cortactin causes shortening and disorganization of those TBCs at the apex of the epithelium that are associated with late spermatids, and this is correlated with a delay in sperm release. However, it has not been shown if disruption of cortactin expression has similar effects on TBCs associated with the basal junction complexes and whether or not this has an effect on junction removal. Here we use electron microscopy (EM) and stimulated emission depletion (STED) super resolution microscopy to evaluate the phenotype of rat testes injected 5 times over a 9‐day period with siRNA targeting cortactin, and use western blots to confirm the knockdown. In vivo knockdown of cortactin in rat testes disrupts basal TBCs when sections labeled for ORP9 and cortactin/actin are evaluated by STED, and leads to the presence of redundant junction related material (ectoplasmic specializations) when evaluated by EM. These data are consistent with the conclusion that TBCs internalize basal junctions and are likely part of the mechanism of spermatocyte translocation from basal to adluminal compartments of the epithelium.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A Wayne Vogl (RGPIN‐2018‐03727).

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca2+) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca2+ oscillations within mammalian eggs. Moreover, studies describing PLCζ “knockout” mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca2 + oscillations and egg activation in mammals, while also discussing the nature of a potential “alternative” sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.

Expression of Taste Receptor 2 Subtypes in Human Testis and Sperm.

Taste receptors (TASRs) are expressed not only in the oral cavity but also throughout the body, thus suggesting that they may play different roles in organ systems beyond the tongue. Recent studies showed the expression of several TASRs in mammalian testis and sperm, indicating an involvement of these receptors in male gametogenesis and fertility. This notion is supported by an impaired reproductive phenotype of mouse carrying targeted deletion of taste receptor genes, as well as by a significant correlation between human semen parameters and specific polymorphisms of taste receptor genes. To better understand the biological and thus clinical significance of these receptors for human reproduction, we analyzed the expression of several members of the TAS2Rs family of bitter receptors in human testis and in ejaculated sperm before and after in vitro selection and capacitation. Our results provide evidence for the expression of TAS2R genes, with TAS2R14 being the most expressed bitter receptor subtype in both testis tissue and sperm cells, respectively. In addition, it was observed that in vitro capacitation significantly affects both the expression and the subcellular localization of these receptors in isolated spermatozoa. Interestingly, α-gustducin and α-transducin, two Gα subunits expressed in taste buds on the tongue, are also expressed in human spermatozoa; moreover, a subcellular redistribution of both G protein α-subunits to different sub-compartments of sperm was registered upon in vitro capacitation. Finally, we shed light on the possible downstream transduction pathway initiated upon taste receptor activation in the male reproductive system. Performing ultrasensitive droplets digital PCR assays to quantify RNA copy numbers of a distinct gene, we found a significant correlation between the expression of TAS2Rs and TRPM5 (r = 0.87), the cation channel involved in bitter but also sweet and umami taste transduction in taste buds on the tongue. Even if further studies are needed to clarify the precise functional role of taste receptors for successful reproduction, the presented findings significantly extend our knowledge of the biological role of TAS2Rs for human male fertility.