Testis Extracts Research Articles

Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity.

Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.

РЕЗУЛЬТАТЫ ЭКСПЕРИМЕНТОВ ПО РАЗРАБОТКЕ ВЫСОКООЧИЩЕННОЙ ГИАЛУРОНАТ-ЭНДО-β-N-АЦЕТИЛГЕКСОЗАМИНИДАЗЫ В КАЧЕСТВЕ ФАРМАЦЕВТИЧЕСКОЙ СУБСТАНЦИИ ДЛЯ ЛЕКАРСТВЕННОГО ПРЕПАРАТА

Enzyme preparations, including hyaluronidase, are widely used in medical practice. Most drugs with this active substance on the pharmaceutical market are produced mainly from testicular hyaluronidase, the raw material for which are bovine testes. The technologies of enzyme preparation production are quite well known and diverse. However, the creation of a different method will allow the highly purified hyaluronate-endo-β-N-acetylhexosaminidase with high specific enzymatic activity. Objective: develop a technology and obtain a laboratory batch of the highly purified pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase. Materials and Methods. In the experimental study, preparations of bovine testes extracts were taken to obtain highly purified hyaluronate-endo-β-N-acetylhexosaminidase. In the first stage the protein spectrum of the testes extracts was studied, proteins were fractionally precipitated, and substances of different chemical nature were fractionated by chromatography methods. At the second stage, the development of a method to obtain the pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase was carried out using the methods described further in the work. Results. Based on the results obtained during the experimental study, P-II, a preparation of bovine testes extract produced by Samson-Med LLC (St. Petersburg), was chosen as the initial substance of crude hyaluronidase for further studies. The most optimal is the purification scheme, which includes as the main method affinity chromatography on heparin-sepharose using affinity elution with heparin solution in buffer with a concentration of 3 mg/ml. Conclusions As a result of the experimental study, the optimal scheme was presented to obtain the pharmaceutical substance HEBNA with high enzymatic total activity of 289 (unit of action) was presented, and a laboratory batch of highly purified hyaluronate-endo-β-N-acetylhexosaminidase was obtained.

Exercise Training and Verbena officinalis L. Affect Pre-Clinical and Histological Parameters.

Verbena officinalis L. or vervain is an herbal medicine and dietary supplement used worldwide. It is used for antidepressant and anticonvulsant purposes, as well as to treat inflammatory disorders, skin burns, abrasions, and gastric diseases, among others. Here, we investigated the biochemical, antioxidant, and histopathological effects of vervain against chronic physical stress. Male Wistar rats were submitted to chronic physical training and oral administration of 200 mg/kg of extract for 7 weeks. Control animals were not treated with either stress or vervain. Body weight was monitored during the study. Liver, kidney, spleen, testis, epididymis, heart, skeletal muscle, and brain samples were collected. Blood cholesterol, lactate dehydrogenase (LDH), bilirubin, and creatinine kinase (CREA), among others, were studied. Glutathione peroxidase (GPox) and superoxide dismutase (SOD) antioxidant activity was analyzed in the blood, liver, and kidney. Testosterone measurements were also performed on whole testis extracts. We found significant weight ratios differences in the epididymis, brain, and heart. Animals submitted to training showed hemorrhagic livers. Kidney histology was affected by both stress and vervain. Cell disruption and vacuolization were observed in the testes and epididymis of animals submitted to stress. Hematological and biochemical markers as CREA, LDH, TP, CKI, URCA, γGT, and glucose revealed statistically significantly differences. Additionally, the activity of glutathione peroxide (GPox) and superoxide dismutase (SOD) in the blood was also impacted. Both stress and vervain have significant in vivo effects. Infusions of vervain include phenylpropanoids, iridoids, verbenalin, hastatoside, and flavonoids, amongst others, which interact synergistically to produce the preclinical effects reported here.

The medicalization of testosterone: reinventing the elixir of life.

The pursuit of longevity, which during the Renaissance era was limited to longing for miraculous ways of rejuvenation, such as bathing in the fountain of youth, took a scientific turn in 1889 with the publication of Brown-Sequard's self-experiments with an extract of animal testes, which apparently improved his vitality, physical strength and cognition. This extract, marketed then as the "Elixir of Life", was sold for decades throughout Europe and North America. However, recent replication of Brown-Sequard's experiments demonstrated that such an extract only contains homeopathic concentrations of testosterone that are insufficient to exert any biological effect. Thus, the birth of Andrology began with a placebo effect. Over the past few decades, the quest for compounds that might lead to rejuvenation has regained traction, with testosterone being at the forefront. Though clinical practice guidelines advocate testosterone therapy in men with organic hypogonadism-the only indication approved by the Food and Drug Administration-testosterone continues to be marketed as a wonder drug with rejuvenating effects on sexual function, vitality, and a host of other unproven benefits. Additionally, the epidemic of obesity and diabetes, conditions associated with low testosterone, has further brought testosterone into the limelight. Although the number of testosterone prescriptions written have increased several-fold in the past two decades, carefully conducted randomized trials suggest modest benefits of testosterone therapy. At the same time, safety concerns, particularly in older men, remain valid.

Regulation of macrophage number and gene transcript levels by activin A and its binding protein, follistatin, in the testes of adult mice

The cytokine activin A is expressed throughout testicular development and is a critical regulator of macrophage function, but its effects on the testicular macrophages are not well-defined. Macrophage distribution and gene transcript levels were examined in testes of adult mice with reduced levels of either activin A (Inhba+/-), or its binding protein, follistatin (TghFST315). Macrophages were identified using F4/80 immunohistochemistry and enumerated by morphometry. Transcript levels were measured in testis extracts by qRT-PCR and Fluidigm ™ analyses. Interstitial macrophages were twice as numerous as peritubular macrophages in Inhba+/- and TghFST315 mice and their littermate controls. Macrophage numbers were significantly reduced in all regions of the Inhba+/- testis, and the volume density of peritubular and subcapsular macrophages was significantly reduced compared to littermate controls (by 52.9% and 36.3% respectively). Transcripts encoding macrophage chemokines, Csf1 and Ccl2, and receptor Csf1r, were elevated (by 35%, 44% and 27% respectively) in Inhba+/- testes, but Cx3cl1 and their receptors, Cx3cr1 and Ccr2, were not altered. Transcripts encoding MHC class II antigens and the co-stimulatory molecule Cd86, also increased (by 32% and 60% respectively), but other co-stimulatory molecules Cd80 and Cd274, and the scavenger receptor Mrc1 (CD206), were unaffected. In the follistatin-deficient testes, macrophage numbers and most macrophage-specific transcripts were not significantly affected, but Mrc1 expression was reduced by 35%. These data indicate that activin A maintains macrophage numbers, but selectively inhibits the levels of key transcripts associated with macrophage antigen-presentation, recruitment and differentiation in the adult mouse testis.

MEIG1 determines the manchette localization of IFT20 and IFT88, two intraflagellar transport components in male germ cells

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.

Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

The concentration of androgen receptor and protein kinase A in male chicken following the administration of a combination of the epididymis and testicular extracts

Background and Aim:Testis (T) and epididymis (E) are waste from the abattoir that is rarely used. In fact, both organs contain important chemicals needed for spermatogenesis (e.g., hormones, proteins, and other molecules). Therefore, administration of a combination of testis and epididymis (CTE) extracts may activate androgen receptors (AR) and protein kinase A (PKA) molecules that play a prominent role in spermatogenesis. We, therefore, aimed at investigating the influence of the CTE extracts on the concentration of AR and PKA in male chicken.Materials and Methods:This study used a completely randomized design with four treatment groups (K0, K1, K2, and K3) and five replications per group. K0 is a control group that received 1 mL normal saline, whereas K1, K2, and K3 are the test groups that received 1, 2, and 3 mL of CET extracts, respectively. Twenty male chickens (strain: broiler Mb 89), 3 weeks of age, weighing 500-700 g were used. We administered the injections in a 13-day period and on the 14th day; we collected and processed blood samples as serum to measure the AR and PKA concentrations using commercial chicken AR and PKA enzyme-linked immunosorbent assay kits, respectively. We performed analyses by analysis of variance using SPSS 20.0.Results:The AR concentrations in K1, K2, and K3 groups increased by 4.26%, 10.97%, and 28.04%, respectively, compared to the K0 (control group). However, this increase was not significantly different between the groups (p>0.05). Moreover, the PKA concentrations increased by 2.97%, 2.60%, and 4.08% in K1, K2, and K3 groups, respectively, compared to the control group. However, this increase was not significantly different between the groups as well (p>0.05).Conclusion:The CTE extracts tended to increase the AR and PKA concentrations even though it is not significant. Therefore, it needs further study when using the CTE extracts for spermatogenesis in male chicken.

Mass spectrometric identification of candidate RNA-binding proteins associated with Transition Nuclear Protein mRNA in the mouse testis

Spermatogenesis is a differentiation process that requires dramatic changes to DNA architecture, a process governed in part by Transition Nuclear Proteins 1 and 2 (TNP1 and TNP2). Translation of Tnp1 and Tnp2 mRNAs is temporally disengaged from their transcription. We hypothesized that RNA regulatory proteins associate specifically with Tnp mRNAs to control the delayed timing of their translation. To identify potential regulatory proteins, we isolated endogenous mRNA/protein complexes from testis extract and identified by mass spectrometry proteins that associated with one or both Tnp transcripts. Five proteins showed strong association with Tnp transcripts but had low signal when Actin mRNA was isolated. We visualized the expression patterns in testis sections of the five proteins and found that each of the proteins was detected in germ cells at the appropriate stages to regulate Tnp RNA expression.

Obtaining and characterization of anti-testis monoclonal antibodies: Invaluable tools toward the identification of testis antigens involved in fertilization.

Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.

Pig testis extract augments adiponectin expression and secretion through the peroxisome proliferator-activated receptor signaling pathway in 3T3-L1 adipocytes.

Adiponectin is a key molecule whose upregulation may ameliorate symptoms of type 2 diabetes mellitus and disorders of lipid metabolism. Several plant-derived components have been shown to enhance adiponectin secretion; however, there have been no reports on the effects of animal-derived products. Therefore, in the current study, we investigated whether hot-water extracts of specific livestock by-products induce the expression of adiponectin in mouse 3T3-L1 adipocytes. Out of the 11 extracts tested, pig testis extract (PTE) was found to enhance adiponectin expression and secretion by 3T3-L1 cells. Furthermore, simultaneous treatment with PTE and daidzein, a soy phytoestrogen, synergistically enhanced adiponectin secretion. Moreover, pretreatment with an estrogen receptor β antagonist (PHTPP) diminished adiponectin secretion from daidzein-treated cells but not from PTE-treated cells. Transcriptome analyses revealed that both daidzein and PTE regulate the peroxisome proliferator-activated receptor signaling pathway, although differences in the regulation of gene expression were observed between PTE- and daidzein-treated cells. These results suggest that PTE ameliorates lipid metabolic dysfunction by promoting adipocyte differentiation and enhancing adiponectin secretion via a mechanism different from that of daidzein.

Masculinization of nile tilapia (Oreochromis niloticus) using extract of bull testes

Tilapia males grow faster to increase the production of the fish, there is a necessity to produce all males by using extract of bull testes. The research method used in this study was the experimental Completely Randomized Design (CRD) with five treatments and three replications. The treatments were A: without immersion and without oral treatment (control); B; immersion 600 μg·L-1 without oral treatment; C: immersion 600 μg·L-1 and oral at a dose of 30 mg·kg-1 feeds, D: immersion 600 μg·L-1 and oral at a dose of 40 mg·kg-1 feeds, E: immersion 600 μg·L-1 and oral at a dose of 50 mg·kg-1 feeds. Results showed that the use of bull testicle extract mixed to media at concentration of 3 ml L-1 produced red male nile of 69.07 %. In addition, the immersion of the extract at 500 μg·L-1 resulted the male of 86.71 %. Sex reversal by dipping at 600 μg·L-1 17α-methyltestosterone combined with oral administration at 40 mg·kg-1 produced the highest male of Nile Tilapia Java carp (86.67 %) compared with dipping, which produced 65.56 % of male. These results suggested that combination of dipping and oral is more effective for sex reversal.

Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

BackgroundHistones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants.ResultsIn this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility.ConclusionsOur results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases.

Testosterone and Male Aging

Hopes for hormonal rejuvenation appear periodically throughout history—with the most prominent attempt occurring around the turn of the 20th century only to vanish in the 1930s following the discovery of testosterone, which discredited testis extracts and manipulations. In recent decades, there has been a renewed attempt for hormonal rejuvenation with testosterone in men. Today, 8 decades since the first clinical use of testosterone,1 the sole unequivocal indication for testosterone treatment is as replacement therapy for men with pathological hypogonadism (ie, organic disorders of the reproductive system).2 Yet despite no proven new indications, global testosterone sales increased 100-fold over the last 3 decades, including increases of 40-fold in Canada and 10-fold in the United States from 2000-2011.3 This was achieved by marketing strategies that circumvented the need for efficacy and safety testing of testosterone for male aging by stretching the definition of the term hypogonadism to encompass virtually any condition associated with low circulating testosterone levels. Promoted under the rubric “low T” (also referred to as andropause or late-onset hypogonadism), this process was facilitated by individual physicians and professional societies that minimized the fundamental distinction between pathological hypogonadism and functional states (including aging) associated with low circulating testosterone levels.

Induction of male germ cell-like lineage from chicken fetal bone marrow stem cells with chicken testis extract

Bone marrow-derived stem cells (BMCs) are able to differentiate into multilineage cells such as muscle, bone, cartilage, fat, and nerve cells. In the present study, we investigated the differentiation capability of chicken BMCs into germ cells by using retinoic acid (RA) and chicken testis extract (chTE). The chicken BMCs were isolated from fetal chicken femurs on post-fertilization day 20, cultured in vitro, and treated with RA and chTE, respectively. The cultured chicken BMCs displayed fibroblast-like morphology and were positive for mesenchymal-specific markers such as CD44, CD90, and CD105 at the mRNA level. RT-PCR and immunocytostaining revealed that both RA and chTE treatments induced the expression of early-germ-cell markers such as Stra8, Dazl and DDX4. The increase of germ cell-specific gene expression after chTE treatment indicates that testicular environment-derived proteins may induce in vitro germ-cells. In addition, we performed a microarray analysis to identify differentially expressed genes (DEGs) in RA and chTE, respectively. A total of 1,629 DEGs were obtained and the chTE treatment showed very lower numbers of DEGs than the RA treatment. Collectively, our results indicate that chicken BMCs have the potential to differentiate to male germ cells in vitro with testis derived proteins.

Mouse DNA polymerase ι lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro

In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a polι peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no polι protein was detected in testes extracts from 129X1/SvJmice, where wild-type polι is normally highly expressed. The early truncation in polι occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in polι activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less polι protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less polι isoform possesses catalytic activity in vitro, we have purified a glutathione-tagged full-length exon-2-less (675 amino acid) polι protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse polι in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse polι exhibited robust characteristic properties previously associated with human polι’s biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less polι enzyme under the same reaction conditions and conclude that exon-2-less polι is indeed rendered catalytically inactive in vitro.

Lactate Dehydrogenase C Produces S-2-Hydroxyglutarate in Mouse Testis.

Metabolomics is a valuable tool for studying tissue- and organism-specific metabolism. In normal mouse testis, we found 70 μM S-2-hydroxyglutarate (2HG), more than 10-fold greater than in other tissues. S-2HG is a competitive inhibitor of α-ketoglutarate-dependent demethylation enzymes and can alter histone or DNA methylation. To identify the source of testis S-2HG, we fractionated testis extracts and identified the fractions that actively produced S-2HG. Through a combination of ion exchange and size exclusion chromatography, we enriched a single active protein, the lactate dehydrogenase isozyme LDHC, which is primarily expressed in testis. At neutral pH, recombinant mouse LDHC rapidly converted both pyruvate into lactate and α-ketoglutarate into S-2HG, whereas recombinant human LDHC only produced lactate. Rapid S-2HG production by LDHC depends on amino acids 100-102 being Met-Val-Ser, a sequence that occurs only in the rodent protein. Other mammalian LDH can also produce some S-2HG, but at acidic pH. Thus, polymorphisms in the Ldhc gene control testis levels of S-2HG, and thereby epigenetics, across mammals.

The impact of sheep testis extract on hair follicle growth and skin healing of Wistar rats

The impact of sheep testis extract on hair follicle growth and skin healing of Wistar rats

Measurement of testosterone by immunoassays and mass spectrometry in mouse serum, testicular, and ovarian extracts.

Accurate measurement of testosterone is important for reproductive endocrinology research, but the validity of direct (nonextraction) testosterone immunoassays, developed and validated for human serum, has not been appraised for application to mouse serum or steroidogenic tissue extracts. Testosterone was measured in serum and extracts of testis or ovary from male and female wild-type mice by 2 commercial direct testosterone immunoassays, with and without preassay extraction, and by the liquid chromatography, tandem mass spectrometry reference method. Results were compared hierarchically by correlation (Kendall's τ), regression (Passing-Bablok), and deviance (Bland-Altman) analysis, under the null hypothesis of perfect agreement between assays (slope = 1, intercept and deviation = 0). For mouse serum, immunoassays displayed an upward bias with performance better for male vs female sera and, within gender, improved by preassay extraction relative to liquid chromatography, tandem mass spectrometry. Testosterone was detectable in all serum samples, but few (male 54%, female 9%) were accurate (within 20% of the reference measurement). For mouse testis extracts, immunoassays were biased upwards, and preassay extraction improved immunoassay performance. Although testosterone was detectable in all extracts, a minority (45%) was accurate. For mouse ovary extracts, all correlations were poor with severe, upward bias, and while testosterone was detectable in all samples, virtually none were accurate. We conclude that these direct testosterone immunoassay kits provide relatively, but not absolutely, accurate results with male mouse serum and testis extracts but not with female mouse serum and ovary extracts, with performance improved by preassay extraction. Whether relative accuracy is fit for purpose depends on the experimental aims, design, and interpretation.

Characterization and expression of trypsinogen and trypsin in medaka testis.

Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.