Testis Extracts Research Articles

Exploring the Role of Rhodtestolin, A Cardio-Inhibitor from the Testes of Rhodnius prolixus, in Relation to the Structure and Function of Reproductive Organs in Insect Vectors of Chagas Disease

Rhodtestolin is a cardio-inhibitor that was first discovered in testes extracts of the blood-feeding insect, Rhodnius prolixus. Its role in reproduction remains unconfirmed, but if delivered to the female during spermatophore formation, it may serve to calm the female and/or relax the vaginal muscles to facilitate delivery and storage of the spermatophore. We describe here the anatomy of reproductive organs in R. prolixus and show that rhodtestolin is present in a low-molecular weight fraction of testes extracts separated by gel filtration, as well as in spermatophores delivered to the female during spermatophore formation. We also report that a rhodtestolin-like factor is present in the testes of R. brethesi, Triatoma dimidiata, T. klugi and Nesotriatoma bruneri, other Reduviidae, which are vectors of Chagas disease. Male secretions in insects are known to modify female behavior after copulation, and the presence of rhodtestolin in several genera of Reduviidae suggests that it plays an important role in reproductive success. Determining this role could lead to developing additional population control strategies for these bugs.

Introduction to the HESI‐Sponsored Inhibin Consortium

The existence of a compound from the tubular fraction of the testis which acts on the pituitary was first proposed in writing by McCullagh in 1932. This was inferred by observing the differential effects on the prostate and the pituitary when animals were administered water-soluble or ether-soluble extracts of testis. Because the water-soluble extract inhibited pituitary hypertrophy, he dubbed this moiety inhibin (his androitin for the ether-soluble actor later became testosterone). The early literature has been nicely reviewed (Ying, 1988). The early assays for inhibin were bioassays (e.g., Steinberger and Steinberger, 1976), later superseded by the more-sensitive radioimmunoassays, and now ELISAs. Inhibin has been used for several years as a clinical tool to help diagnose and categorize male infertility patients (e.g., Pierik et al., 1998; reviewed in Lockwood 2004, Adamopoulos and Koukkou, 2010, and Anderson and Sharpe, 2000). There has been a robust literature on various aspects on the physiology of the hormone, for example, trying to discern what controls circulating levels of Inhibin B in males (e.g., Ramaswamy et al., 1999), or describing the relationship between Inhibin and FSH (e.g., von Eckerdstein et al., 1999; Kamischke et al., 2001), or determining the impact of different germ cell populations on circulating Inhibin B levels (e.g., Pineau et al., 1989; Allenby et al., 1991; Clifton et al., 2002), or outlining Inhibin B’s role in development (Byrd et al., 1998) or evaluating its use as an epidemiologic marker (e.g., Ellingsen et al., 2007). There are numerous excellent reviews on this subject, a few of which are Meachem et al. (2001), de Kretser et al. (2004), and Stewart and Turner (2005). Because the large interand intraindividual variability makes sperm count and semen studies quite insensitive (e.g., Schrader et al., 1988), toxicologists and epidemiologists have long been looking for a more sensitive (i.e., less noisy and more responsive) biomarker of damage to the seminiferous tubules. Inhibin B seemed to fill this need, and indeed was found to respond to several different types of toxicities, especially over sufficient periods of time (Wallace et al., 1997; Tsatsoulis et al., 1988; Foppiani et al., 1999; Hild et al., 2001; Suescun et al., 2001; rev. in Stewart and Turner, 2005; Tunc et al., 2006).

RABL-regulated pathways: a new tale in sperm function

RABL-regulated pathways: a new tale in sperm function

Effects of porcine testis extract on wound healing in rat

Sex hormones have long been considered to play an important role in bone turnover rate, periodontal diseases, and wound healing. We have studied the effect of porcine testis steroid extract (PTSE), an extract of porcine testes, which holds a good ratio of 19-nortestosterone (nandrolone), testosterone, androstenedione, 17β-estradiol, and estrone, on the healing rate of a standardized full-thickness linear wound on the back of the rat. Skin punch or carbon dioxide (CO2) laser methods were used to create the deep skin injury in two groups of animals. The animals were treated with the PTSE cream, control cream and Vaseline (control) to find out the effect in re-epithelialization, contraction, and formation of granulation and scar tissues. Histological examination after 21 days showed 100, 87.4, and 80.5% recovery of epidermis, dermis, and hypodermis, respectively in the PTSE-treated animals. Similarly, on the 15th day of treatment, complete healing of intact skin was observed in the PTSE cream-treated animals among the laser radiation group. Even though the beginning of re-epithelialization phase and completion of serum crust formation was also observed in the base cream- and Vaseline-treated animals respectively, the complete healing cycle was observed only in the PTSE-treated group. The white blood cell count in the PTSE-treated group showed that PTSE cream is nontoxic to animals.

Liprin α3: a putative estrogen regulated acrosomal protein

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.

The Expression of Tubulin Cofactor A (TBCA) Is Regulated by a Noncoding Antisense Tbca RNA during Testis Maturation

BackgroundRecently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules.Methodology/Principal FindingsWe found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line.Conclusions/SignificanceOur results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

Purification and characterization of an antimicrobial histone H1-like protein and its gene from the testes of olive flounder, Paralichthys olivaceus

An approximately 21 kDa antimicrobial protein was purified from an acidified testis extract of olive flounder, Paralichthys olivaceus, by ion-exchange and C18 reversed-phase HPLC. A comparison of the N-terminal amino acid sequence with those of other known antimicrobial polypeptides revealed high homology between this antimicrobial protein and other histone H1 molecules; thus, it was designated flounder histone H1-like protein (fH1LP). fH1LP showed potent antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 2.8–30.0 μg/ml), Gram-negative bacteria, including Aeromonas hydrophila, Escherichia coli D31, Vibrio parahaemolyticus (MECs, 1.4–12.0 μg/ml), and Candida albicans (MEC, 2.0 μg/ml). cDNA cloning and tissue distribution studies of fH1LP indicated that it is constitutively expressed in testis and ovary. The fH1LP expression level was significantly dependent on developmental stage, and decreased dramatically after hatching. However, lipopolysaccharide stimulation did not induce fH1LP mRNA in other immune organs, including the kidney and spleen. These results suggest that fH1LP plays an important role in innate immunity in fish during reproduction, including mating, fertilization, and hatching.

A novel testis‐specific isoform of the protein phosphatase1 inhibitor I‐2 (Ppp1r2) is expressed in developing germ cells during sperm morphogenesis.

The protein Inhibitor‐2 (I‐2, PPP1R2) of the serine/threonine phosphatase PP1 is one of the ancient PP1 binding proteins. An isoform of PP1, PP1γ2, is highly expressed in testis. This isoform, present only in mammals, is essential for spermatogenesis and sperm function. The coding sequence and the protein product of this message were not identified. We now show that this 0.9 kb message is expressed in developing germ cells during differentiation of haploid spermatids into spermatozoa. Using a spermatid cDNA library and RT‐PCR of testis RNA we have shown that the 0.9 kb mRNA arises due to retention of the last intron (intron 4) that is spliced out in the somatic form of I‐2 mRNA. This intron retention leads to the truncated message that generates a protein with a unique C‐terminus not found in the somatic cell form of I‐2. Protein sequence analysis using MS of heat stable testis extracts confirmed the amino acid sequence of the unique germ cell form of I‐2. Remarkably two other protein regulators of sperm PP1γ2, sds22 and I‐3, are also expressed as testis specific isoforms during late stages of spermatogenesis. Studies are underway to determine the significance of high levels of expression of testis specific versions of these PP1 regulators in spermatogenesis and sperm function. (NIH HD38520)

Cancer associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.

Identification of the estrogen receptor GPER in neoplastic and non-neoplastic human testes.

BackgroundEstrogen signaling is mediated by estrogen receptor beta isoforms in normal and neoplastic human testes. Recently, a G-protein-coupled-receptor (GPER) has been suggested as being involved in rapid responses to estrogens in different normal and tumor cells.MethodsThis study investigated the GPER expression in paraffin-embedded samples from non neoplastic and neoplastic human testes (sex-cord stromal and germ cell tumors) by immunohistochemical and Western Blot analyses.ResultsIn control testes, a positive GPER immunoreactivity was detected in Leydig and in Sertoli cells while all germ cells were immunonegative. Furthermore, neoplastic cells of the Sertoli cell tumor, Leydig cell tumor, seminoma and embryonal carcinoma samples were all immunopositive. The immunoblots of testis extracts confirmed the results.ConclusionsThese findings suggest that GPER could mediate estrogen signaling in both normal and transformed somatic cells of human testis, but they reveal a differential expression of the novel estrogen receptor in non neoplastic and neoplastic germ cells.

Proteolytically Activated, Recombinant Anti-Müllerian Hormone Inhibits Androgen Secretion, Proliferation, and Differentiation of Spermatogonia in Adult Zebrafish Testis Organ Cultures

Anti-Müllerian hormone (Amh) is in mammals known as a TGFβ type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis.

Molecular Recognition of the Tes LIM2–3 Domains by the Actin-related Protein Arp7A

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized “orphan” Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1–65Arp7A·LIM2–3Tes·EVH1Mena complex reveals that residues 28–49 of Arp7A contact the LIM2–3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2–3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated ‘rpL22-like short’) that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.

Sohlh1 and Sohlh2 Double Knockouts Reveal Cooperative Function in the Male and Female Germ Cell Differentiation Pathways.

Proper maintenance of germ cells and their differentiation are essential determinants of reproductive life. We discovered two novel germ cell specific transcriptional regulators, Sohlh1 and Sohlh2. Sohlh1 and Sohlh2 encode basic helix-loop-helix transcriptional regulators and are essential in early folliculogenesis and spermatogonial differentiation, as shown by individual knockouts. Sohlh1 and Sohlh2 individual knockouts show remarkably similar phenotypes. Here we examined the phenotype and gene expression in mice that lack both Sohlh1 and Sohlh2 transcriptional regulators. Antibodies against Sohlh1 and Sohlh2 show similar pattern of expression in both male and female germ cells. In males, Sohlh1 and Sohlh2 are expressed in spermatogonia, and co-expressed with Plzf and c-Kit. In females, antibodies against Sohlh1 and Sohlh2 label the same population of oocytes. In order to determine whether Sohlh1 and Sohlh2 physically interact, we utilized testes extracts to show that Sohlh1 and Sohlh2 co-immunoprecipitate, sugessting that Sohlh1 and Sohlh2 heterodimerize. In addition, Sohlh2 expression is not completely abrogated in Sohlh1-/- mice and vice versa. These data imply that residual Sohlh1 and Sohlh2 proteins were produced in each others mutant, possibly alleviating a more severe phenotype. We therefore analyzed Sohlh1-/-Sohlh2-/- double knockout mice. Sohlh1-/-Sohlh2-/- newborn ovary retains as many oocytes as wild-type. By 3-week of age, the double mutant ovary contains few if any oocytes as single mutants. We also analyzed testes development in double mutants. In postnatal day 7 double mutant testes, there is no obvious difference as compared to the wild type but spermatogonial differentiation is disrupted at 2-week of age and beyond, a finding similar to single gene mutants. The spermatogonias of single or double mutant retain Plzf expression, and continue to proliferate as shown by BrdU incorporation. Thus histological analysis revealed that male and female double mutants are a phenocopy of Sohlh1-/- and Sohlh2-/-. However, microarray analysis of double mutant and single mutants show a synergistic effect of the combined Sohlh1 and Sohlh2 deficiency on gene expression. In testis, although there is statistically no significant difference between Sohlh1-/- and Sohlh2-/-, Sohlh1-/-Sohlh2-/- versus wild-type shows nearly 4-fold probe change as compared to the case of single mutants versus wild-type. These studies indicate that Sohlh1 and Sohlh2 are purely essential for oocyte and spermatogonia differentiation, and function either cooperatively or independently in those pathways. (platform)

Co‐purification of Mac‐2 binding protein with galectin‐3 and association with prostasomes in human semen

Prostasomes are exosome-like vesicles that are secreted by the prostate and incorporated into semen during ejaculation. Human prostasomes are proposed to function in regulation of sperm function, immunosuppression, and prostate cancer progression. Previously, we identified galectin-3 on the surface of prostasomes. Galectin-3 is a β-galactoside binding protein involved in immunomodulation, cell interactions, and cancer progression, including prostate cancer. Functional characterization of galectin-3 in a given biological environment includes identification of its target glycoprotein ligands. Candidate galectin-3 ligands in prostasomes were identified by tandem mass spectrometry of proteins that co-purified with galectin-3 during lactose affinity chromatography. Immunochemical and biochemical methods were used to investigate the association of Mac-2 binding protein (M2BP) with prostasomes. Proteins identified by tandem mass spectrometry included M2BP, CD26/dipeptidyl peptidase IV, prolactin-inducible protein (PIP), olfactomedin-4 (OLFM4), and semenogelins I and II (SgI and SgII). M2BP is a known galectin-3 ligand that was not previously described in prostasomes. M2BP protein bands were detected in the testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm extracts. In seminal plasma, M2BP was identified in the soluble fraction and in purified prostasomes. Surface biotinylation and immunofluorescence studies indicated that M2BP is present on the prostasome surface and on sperm, respectively. M2BP, CD26, PIP, OLFM4, and SgI and SgII are candidate glycoprotein ligands for galectin-3 in prostasomes. Given their overlap in functional significance with prostasomes and galectin-3, the identification of these glycoproteins as galectin-3 ligands in prostasomes lays the groundwork for future studies of prostasomes in reproduction and prostate cancer.

Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3kDa; fraction II, 3–14kDa; and fraction III, >14kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.

Testes extracts inhibit heart contractions in females of the blood‐feeding insect, Rhodnius prolixus

Abstract Myogenic contractions of the heart of the female blood‐feeding insect, Rhodnius prolixus (Stål), are inhibited by crude extracts of testes applied directly to isolated dorsal vessels. Dorsal vessels were observed with a stereo microscope and heart beats timed with a stopwatch. In normal Rhodnius saline, hearts contract at 14.8 ± 7.1 beats per minute (n= 45). Crude extracts of the testes and the two male reproductive accessory organs (the opaque and transparent accessory glands) were prepared from previously frozen tissue by homogenizing 5–20 glands in a small glass homogenizer containing Rhodnius saline, centrifuging for 5 min at 2 000 g, and collecting the supernatant. Testes extract as low as 1.0 glands per mL inhibit contractions whereas crude extracts of the opaque or transparent accessory glands have no consistent effect. We refer to this cardiac inhibitor as rhodtestolin (Rhodnius testis inhibitory factor), and discuss its possible effects on the female during copulation.

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

Mammalian glutamate dehydrogenase (GDH) is an allosterically regulated enzyme that is expressed widely. Its activity is potently inhibited by GTP and thought to be controlled by the need of the cell for ATP. In addition to this housekeeping human (h) GDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. Although transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here, we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed, to our surprise, that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immunocytochemical and immunofluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immunoreactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the tricarboxylic acid cycle via conversion of glutamate to alpha-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the tricarboxylic acid cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1 protein. Hence, the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells.

Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase

Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at approximately 5 pm and nadir at approximately 5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.