Daniel G. Cyr

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: Background to spermatogenesis, spermatogonia, and spermatocytes

Spermatogenesis, a study of germ cell development, is a long, orderly, and well‐defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well‐defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (As), proliferating cells (Apr, Aal), and differentiating cells (A1–A4, In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event. Microsc. Res. Tech., 2010.

Contaminant effects on the teleost fish thyroid.

Numerous environmentally relevant chemicals, including polychlorinated hydrocarbons, polycyclic aromatic hydrocarbons, organochlorine pesticides, chlorinated paraffins, organophosphorous pesticides, carbamate pesticides, cyanide compounds, methyl bromide, phenols, ammonia, metals, acid loads, sex steroids, and pharmaceuticals, exert acute or chronic effects on the thyroid cascade in the approximately 40 teleost fish species tested to date. Thyroid endpoints, therefore, serve as biomarkers of exposure to environmental pollutants. However, the mechanisms underlying thyroid changes and their physiological consequences are poorly understood because the thyroid cascade may respond indirectly and it has considerable capacity to compensate for abuses that otherwise would disrupt thyroid hormone homeostasis. Indeed, a xenobiotic-induced change in fish thyroid function has yet to be conclusively causally linked to decreased fitness or survival. Other complications in interpretation arise from the diversity of test conditions employed and the often indiscriminate use of numerous thyroid endpoints. Future work should be directed toward standardizing test conditions and thyroid endpoints and investigating causal links between thyroid changes and fish growth, reproduction, and development. Development may be particularly susceptible to thyroid disruption, and thyroid endpoints appropriate for early life stages need to be applied.

The Role of Mitochondrial DNA Copy Number in Mammalian Fertility

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000–50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

Proliferation of Adult Sertoli Cells Following Conditional Knockout of the Gap Junctional Protein GJA1 (Connexin 43) in Mice

Abstract GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals

The effectiveness of toxicology biomonitoring programs could be improved by the addition of sensitive biomarkers. In this study the cell viability and sensitivity of phagocytic function of phagocytes from bivalves (Mya arenaria) to selected heavy metals were measured by flow cytometry, a novel approach. Hemocytes (phagocytes) collected from bivalves by puncture of the posterior adductor muscle were incubated in vitro for 18 h in hemolymph containing 10(-9)-10(-3)M of cadmium chloride, zinc chloride, mercuric chloride, methylmercury chloride or silver nitrate, before determining their capacity to phagocytose fluorescent latex beads by flow cytometry. Heterogeneity of the hemocyte cell population was determined by forward scatter (FSC) and side scatter (SSC) cytometric profile which showed two distinct cell populations. At low doses (10(-9), 10(-8) M), all the metal compounds studied stimulated phagocytic activity except silver nitrate. At higher levels of exposure (10(-6), 10(7) M), all metals caused a significant concentration-related decrease in hemocyte phagocytosis activity. From the concentration of each metal inducing 50% suppression (IC50) of the phagocytic activity, the immunotoxic potential of metals with respect to phagocytic function can be ranked in the following increasing order: ZnCl2 < CdCl2 < AgNO3 < HgCl2 < CH3HgCl. Parallel analysis of hemocyte viability showed that suppression of phagocytosis by heavy metals was not solely related to a decreased cell viability. These results reveal the high but different degree of sensitivity of the phagocytosis activity of bivalves with respect to heavy metals, as measured by flow cytometry, and demonstrate that flow cytometry is a potentially useful tool in ecotoxicological monitoring.

Phagocytosis as a Biomarker of Immunotoxicity in Wildlife Species Exposed to Environmental Xenobiotics

*Institut National de la Recherche Scientifique-Institut Armand-Frappier (INRS-IAF), Pointe-Claire, QC H9R 1G6, Canada †Departement of Fisheries and Oceans, Institut Maurice-Lamontagne, Mont-Joli, QC G5H 3Z4, Canada ‡Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada §Biomedical Sciences, Ontario Veterinary College Medicine, Guelph, ON N1G 2W1, Canada ¶Groupe de Recherche en Environnement Côtier, Université du Québec à Rimouski, Rimouski, QC G5L 3A1, Canada

Claudin-1 Is Not Restricted to Tight Junctions in the Rat Epididymis1

The blood-epididymal barrier creates a unique microenvironment critical for sperm maturation. There is little information on proteins comprising epididymal tight and adhering junctions or on factors regulating their expression. Claudins are a family of transmembrane proteins reported to be exclusively localized to tight junctions. In the present study the expression of claudin-l (Cl-1) was examined with respect to the different cell types of the epididymis and its various regions as well as its expression during postnatal development and regulation by testicular factors, using both immunocytochemistry and Northern blot analysis. RT-PCR of adult epididymal and testicular RNA (positive control) indicated that Cl-1 messenger RNA (mRNA) transcripts were present in all regions of the epididymis. In the adult, Cl-1 was localized immunocytochemically along the entire length of the lateral plasma membranes between adjacent principal cells, including apical areas containing tight junctions, as well as at the inter...

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 2: Changes in spermatid organelles associated with development of spermatozoa

Spermiogenesis is a long process whereby haploid spermatids derived from the meiotic divisions of spermatocytes undergo metamorphosis into spermatozoa. It is subdivided into distinct steps with 19 being identified in rats, 16 in mouse and 8 in humans. Spermiogenesis extends over 22.7 days in rats and 21.6 days in humans. In this part, we review several key events that take place during the development of spermatids from a structural and functional point of view. During early spermiogenesis, the Golgi apparatus forms the acrosome, a lysosome‐like membrane bound organelle involved in fertilization. The endoplasmic reticulum undergoes several topographical and structural modifications including the formation of the radial body and annulate lamellae. The chromatoid body is fully developed and undergoes structural and functional modifications at this time. It is suspected to be involved in RNA storing and processing. The shape of the spermatid head undergoes extensive structural changes that are species‐specific, and the nuclear chromatin becomes compacted to accommodate the stream‐lined appearance of the sperm head. Microtubules become organized to form a curtain or manchette that associates with spermatids at specific steps of their development. It is involved in maintenance of the sperm head shape and trafficking of proteins in the spermatid cytoplasm. During spermiogenesis, many genes/proteins have been implicated in the diverse dynamic events occurring at this time of development of germ cells and the absence of some of these have been shown to result in subfertility or infertility. Microsc. Res. Tech., 2010.

Claudin-1 is a p63 target gene with a crucial role in epithelial development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias.

Gene Expression Profiling and Its Relevance to the Blood-Epididymal Barrier in the Human Epididymis

Abstract The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.