Testis Extracts Research Articles

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-two-hybrid screen identified the testis-specific kinase, TSSK-2, to be a potential SPAG16L binding partner. SPAG16L and TSSK-2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and from cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK-2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK-2. SPAG16L can be phosphorylated by TSSK-2 in vitro. Finally, TSSK-2 is absent from the testes in approximately half of SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK-2 substrate.

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

Phosphorylation of Mouse Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-Specific Kinase, TSSK21

The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

Germ cell nuclear factor directly represses the transcription of peroxisome proliferator-activated receptor delta gene

Germ cell nuclear factor (GCNF) is a transcription factor that can repress gene transcription and plays an important role during spermatogenesis. Peroxisome proliferator-activated receptor delta (PPARdelta) is a nuclear hormone receptor belonging to the steroid receptor superfamily. It can activate the expression of many genes, including those involved in lipid metabolism. In this report, we showed that GCNF specifically interacts with PPARdelta promoter. Overexpression of GCNF in African green monkey SV40-transformed kidney fibroblast COS7 cells and mouse embryo fibroblast NIH 3T3 cells represses the activity of PPARdelta promoter. The mutation of GCNF response element in PPARdelta promoter relieves the repression in NIH 3T3 cells and mouse testis. Moreover, we showed that GCNF in nuclear extracts of mouse testis is able to bind to PPARdelta promoter directly. We also found that GCNF and PPARdelta mRNA were expressed with different patterns in mouse testis by in situ hybridization. These results suggested that GCNF might be a negative regulator of PPARdelta gene expression through its direct interaction with PPARdelta promoter in mouse testis.

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse

BackgroundThe hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.MethodsInitially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation.ResultsWestern blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on this tissue, and did not augment the actions of estradiol.ConclusionAs ERalpha receptor is abundantly expressed in the pituitary gland of hpg mice, and estradiol did not exert effects on testis development over and above those of FSH, we conclude that the action of estradiol on testis development in hpg mice is predominantly via the stimulation of pituitary FSH release.

Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis

The effects of male-derived extracts on female receptivity to remating were investigated in Callosobruchus chinensis (Coleoptera: Bruchidae). Injection of aqueous extracts of male reproductive tracts into the abdomen of females reduced receptivity. When aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: <3, 3–14, and >14kDa, the filtrate containing MW substances <3kDa reduced female receptivity 3h and 1 day after injection, whereas the fraction containing MW substances >14kDa inhibited receptivity 2 and 4 days after injection. Finally, male reproductive tract organs were divided into accessory gland, seminal vesicle, and testis. Aqueous extracts of testis reduced receptivity of females on the second day and at 3h, and aqueous extracts of accessory gland reduced receptivity of females on the second day after injection. On the other hand, aqueous extracts of seminal vesicle did not reduce female receptivity. The results indicate that more than one mechanism may be involved in producing the effects of male-derived substances on female receptivity; low MW male-derived substances, which possibly exist in testis, cause short-term inhibition, while high MW substances, which possibly exist in the accessory gland, inhibit female mating later than low MW substances in C. chinensis.

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.

Polypyrimidine Tract Binding Protein 2 Stabilizes Phosphoglycerate Kinase 2 mRNA in Murine Male Germ Cells by Binding to Its 3′UTR1

The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3'-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3'-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3'-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.

Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA

Background:The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences.Results:We used a random oligonucleotide in vitro selection method to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence we determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. We also find that some DMRT proteins can bind DNA as heterodimers.Conclusion:Our results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers.

Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-β Results in Lowered Plasma Cholesterol and Elevated Glucagon

Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-β Results in Lowered Plasma Cholesterol and Elevated Glucagon

D-Aspartic acid and nitric oxide as regulators of androgen production in boar testis

d-Aspartic acid ( d-Asp) and nitric oxide (NO) are two biologically active molecules playing important functions as neurotrasmitters and neuromodulators of nerve impulse and as regulators of hormone production by endocrine organs. We studied the occurrence of d-Asp and NO as well as their effects on testosterone synthesis in the testis of boar. This model was chosen for our investigations because it contains more Leydig cells than other mammals. Indirect immunofluorescence applied to cryostat sections was used to evaluate the co-localization of d-Asp and of the enzyme nitric oxide synthase (NOS) in the same Leydig cells. d-Asp and NOS often co-existed in the same Leydig cells and were found, separately, in many other testicular cytotypes. d-Asp level was dosed by an enzymatic method performed on boar testis extracts and was 40 ± 3.6 nmol/g of fresh tissue. NO measurement was carried out using a biochemical method by NOS activity determination and expressed as quantity of nitrites produced: it was 155.25 ± 21.9 nmol/mg of tissue. The effects of the two molecules on steroid hormone production were evaluated by incubating testis homogenates, respectively with or without d-Asp and/or the NO-donor l-arginine ( l-Arg). After incubation, the testosterone presence was measured by immunoenzymatic assay (EIA). These in vitro experiments showed that the addition of d-Asp to incubated testicular homogenates significantly increased testosterone concentration, whereas the addition of l-Arg decreased the hormone production. Moreover, the inclusion of l-Arg to an incubation medium of testicular homogenates with added d-Asp, completely inhibited the stimulating effects of this enantiomer. Our results suggest an autocrine action of both d-Asp and NO on the steroidogenetic activity of the Leydig cell.

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa–egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

Endocannabinoid Biosynthesis Proceeding through Glycerophospho-N-acyl Ethanolamine and a Role for α/β-Hydrolase 4 in This Pathway

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis

Glial cell-line-derived neurotropic factor (GDNF) and its receptors glial cell-line-derived neurotropic factor alpha (GFR1alpha) and rearranged during transformation (RET) have been localized in the rat testis during postnatal development. The three mRNAs, and GDNF and GFR1alpha proteins were detected in testis extracts from 1- to 90-day-old rats by reverse transcriptase PCR and Western blotting respectively. The three mRNAs were present in Sertoli cells from 20- and 55-day-old rats, pachytene spermatocytes (PS), and round spermatids (RS). The GDNF and GFR1alpha proteins were detected in PS, RS, and Sertoli cells. GDNF and GFR1alpha were also detected using flow cytometry in spermatogonia and preleptotene spermatocytes, and in secondary spermatocytes. The localization of GDNF and GFR1alpha in germ and Sertoli cells was confirmed by immunocytochemistry. The hypothesis that GDNF may control DNA synthesis of Sertoli cells and/or spermatogonia in the immature rat was addressed using cultures of seminiferous tubules from 7- to 8-day-old rats. Addition of GDNF for 48 h resulted in a twofold decrease in the percentage of spermatogonia able to duplicate DNA, whereas Sertoli cells were not affected. These results are consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia. Moreover, the wide temporal and spatial expression of GDNF and its receptors in the rat testis suggest that it might act at several stages of spermatogenesis.

Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated

Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein synthesis occurs on polysomes and translationally inactive mRNAs are sequestered as ribonucleoproteins (RNPs), movement of mRNAs between these fractions is indicative of translational up- and down-regulation. Here, we use microarrays to analyze mRNAs in RNPs and polysomes from testis extracts of prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds. Consistent with published reports, many of the translationally delayed postmeiotic mRNAs shift from the RNPs into the polysomes, establishing the validity of this approach. In addition, we detect another 742 mouse testicular transcripts that show dramatic shifts between RNPs and polysomes. One subgroup of 35 genes containing the known, translationally delayed phosphoglycerate kinase 2 (Pgk2) is initially transcribed during meiosis and is translated in later-stage cells. Another subgroup of 82 meiotically expressed genes is translationally down-regulated late in spermatogenesis. This high-throughput approach defines the changing translation patterns of populations of genes as male germ cells differentiate and identifies groups of meiotic transcripts that are translationally up- and down-regulated.

An antibody to the C‐terminus extension of cox2 in the male transmitted mitochondrial genome of Venustaconcha ellipsiformis (Bivalvia: Unionoidea)

Doubly uniparental inheritance of mitochondrial DNA (DUI) has been observed in two orders of marine bivalves and in the freshwater bivalve superfamily Unionoidea. Cox2 is an inner mitochondrial membrane protein that functions in complex IV of the electron transport system. PCR amplification of the cox2-cox1 junction region from unionoidean male-transmitted (M) mtDNA genomes reveals an approximately 555 bp 3′ coding extension to cox2. The fixed presence of the cox2 3′ extension in unionoidean M genomes may be associated with the high stability of DUI in this taxon. We made an antibody against the M cox2 C-terminus extension from V. ellipsiformis. The antibody was characterized by blotting procedures using both soluble and insoluble extracts of testes. Attempts at detecting the lengthened cox2 protein were made using both male and female tissues and it was observed that the protein was predominantly expressed in testes. Weak expression was detected in other male tissues such as foot, mantle and gill. However, female tissues showed no immunoreactive band. Immunohistochemistry also verified that the expression of this protein is predominantly in male spermatogenic tissue. This is the first report documenting the expression of a significantly lengthened cox2 protein in the Unionoidea. We hypothesize that the expression of a lengthened cox2 protein in unionoidean bivalve testes has functional significance.

A false note of DNA polymerase iota in the choir of genome caretakers in mammals

DNA polymerase iota (Pol iota) of mammals is a member of the Y family of DNA polymerases. Among many other genome caretakers, these enzymes are responsible for maintaining genome stability. The members of the Y-family DNA polymerases take part in translesion DNA synthesis, bypassing some DNA lesions, and are characterized by low fidelity of DNA synthesis. A unique ability of Pol iota to predominantly incorporate G opposite T allowed us to identify the product of this enzyme among those synthesized by other DNA polymerases. This product can be called a "false note" of Pol iota. We measured the enzyme activity of Pol iota in crude extracts of cells from different organs of five inbred strains of mice (N3H/Sn, 101/H, C57BL/6, BALB/c, 129/J) that differed in a number of parameters. The "false note" of Pol iota was clearly sounding only in the extracts of testis and brain cells from four analyzed strains: N3H/Sn, 101/H, C57BL/6, BALB/c. In mice of 129/J strain that had a nonsense mutation in the second exon of the pol iota gene, the Pol iota activity was reliably detectable only in the extracts of brain. The data show that the active enzyme can be formed in some cell types even if they carry a nonsense mutation in the pol iota gene. This supports tissue-specific regulation of pol iota gene expression through alternative splicing. A semiquantitative determination of pol iota activity in mice strains different in their radiosensitivity suggests a reciprocal correlation between the enzyme activity of pol iota in testis and the resistance of mice to radiation.

Endogenous testicular D-aspartic acid regulates gonadal aromatase activity in boar

D-aspartic acid (D-Asp), aromatase enzyme activity and the putative D-Asp involvement on aromatase induction have been studied in the testis of mature boars. The peroxidase-antiperoxidase and the indirect immunofluorescence methods, applied to cryostat and paraffin sections, were used to evaluate D-Asp and aromatase distributions. D-Asp level was dosed by an enzymatic method performed on boar testis extracts. Biochemical aromatase activity was determined by in vitro experiments carried out on testis extracts. D-Asp immunoreactivity was found in Leydig cells, and, to a lesser extent, in germ cells. Analogously, aromatase immunoreactivity was present in Leydig cells, but absent from seminiferous tubule elements. In vitro experiments showed that the addition of D-Asp to testicular tissue acetone powder induced a significant increase of aromatase activity, as assessed by testosterone conversion to 17beta-estradiol. Enzyme Km was not affected by D-Asp (about 25 nM in control and D-Asp added tests). These findings suggest that D-Asp could be involved in the local regulation of aromatase in boar Leydig cells and intervenes in this organ's production of estrogens.

143 DEVELOPMENTAL EFFICIENCY IMPROVEMENTS OF NUCLEAR TRANSFER EMBRYOS BY REPROGRAMMING SOMATIC CELLS USING TESTIS-DERIVED CELL EXTRACTS

Somatic cell cloning has promise for medical treatment using embryonic stem cells derived from cloned embryos. However, porcine cloning by somatic cell nuclear transfer has been inefficient and, even after birth, cloned pigs are found to carry a variety of abnormalities. Moreover, recent molecular analyses of cloned embryos have revealed abnormal epigenetic modifications. Therefore, the prevention of epigenetic errors is expected to lead to the improvement of the success rate in animal cloning. Reports of recent studies indicate that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. Therefore, in this study, we modulated the cell fate of somatic donor cells by introducing cell extracts derived from porcine testis. Several porcine somatic cells, including primary and stabilized porcine fibroblasts or epithelial kidney cells, were treated with streptolysin O (SLO; 230 ng mL-1), which reversibly permeablizes plasma membrane, and incubated for 30 min with testis cell-derived cell extracts (4 mg mL-1). To reseal plasma membranes, cells were placed in DMEM containing 30% FBS and 2 mM CaCl2 for 30 min. After resealing the cell membranes, we incubated the cells for 3 weeks and analyzed the expression of testis-specific genes such as protamine 1, protamine 2, SOX 9, mullerian inhibitory substance (MIS), preproacrosine (ACR), phosphoglycerate kinase 2 (PGK-2), protein C, and c-kit ligand. In the reprogrammed primary porcine fibroblasts or epithelial kidney cells, the porcine testis extracts were able to activate the expression of the porcine testis sertoli cell-specific genes. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Then, in vitro-matured oocytes were enucleated and a single cell (either reprogrammed or intact) was injected directly into cytoplasm of the oocytes. The reconstructed embryos were activated electrically and cultured in vitro for 7 days. The rate of blastocyst formation was significantly higher (P &amp;lt; 0.05; chi-square test) in the reprogrammed nuclear donor cells (27/119; 22.7 � 5.0%) than in the control (intact) cells (11/83; 13.3 � 3.2%). Taken together, our results suggest that testis-derived cell extracts can be successfully used to reprogram fibroblasts to express male germ cell function, thus improving the developmental efficiency of the nuclear transfer embryos.