Adult Pig Testis Research Articles

An Epigenome-Wide DNA Methylation Map of Testis in Pigs for Study of Complex Traits.

Epigenetic changes are important for understanding complex trait variation and inheritance in pigs that are also a valuable biomedical model for human health research. Testis is the main organ for reproduction and boar taint in pigs; however, there have been no studies to-date on adult pig testis epigenome. The main objective of this study was to establish a genome-wide DNA methylation map of pig testis that would help identify candidate epigenetic biomarkers and methylated genes for complex traits such as male reproduction, fertility or boar taint. Reduced Representation Bisulfite Sequencing (RRBS) was used to study methylation levels of cytosine in nine pig testis samples. The results showed that genome-wide methylation status of nine samples overlapped greatly and their variation among pigs were low. The methylation levels of promoter, exon, intron, cytosine and guanine dinucleotide (CpG) islands and CpG island shores regions were 0.15, 0.47, 0.55, 0.39, and 0.53, respectively. Cytosines binding to CpG islands showed different methylation levels between exon and intron regions. All methylation levels of CpG islands were lower than CpG island shores in different genic features. The distribution of 12,738 differentially methylated cytosines (DMCs) within CpG islands, CpG island shores and other regions was 36.86, 21.65, and 41.49%, respectively, and was 0.33, 1.71, 5.95, and 92.01% in promoter, exon, intron and intergenic regions, respectively. Methylation levels of DMCs in promoter, exon and intron regions were significantly different between CpG islands and CpG island shores (P < 0.05). A total of 898 genes with 2089 DMCs were enriched in 112 Gene Ontology (GO) terms. Fifteen methylated genes from our study were associated with fertility or boar taint traits. Our analysis revealed the methylation patterns in different genic features and CpG island regions of testis in pigs, and summarized several candidate genes associated with DMCs and the involved GO terms. These findings are helpful to understand the relationship between DNA methylation and genic CpG islands, to provide candidate epigenetic regions or biomarkers for pig production and welfare and for translational epigenomic studies that use pigs as an animal model for human research.

Histone lysine methylation exhibits a distinct distribution during spermatogenesis in pigs

Spermatogenesis is a continual process throughout the adult life of a male, which is governed by unique transcriptional regulation and massive alterations of chromatin. Histone modification was one of the underlying epigenetic mechanisms during spermatogenesis. It has been shown that methylation of histone lysine exhibits a distinct distribution in mice during spermatogenesis and some histone lysine methylation is essential for male fertility. However, the dynamic change of methylated histone in porcine testis tissue was largely unknown. Here, we studied the dynamic modulation of three types of methylation (monomethylation, dimethylation, and trimethylation) of H3K4, H3K27, and H4K20 during spermatogenesis in pigs. The results showed that H3K4me2/3, H3K27me3, and H4K20me1/2/3 were extensively localized in adult pig testis. Interestingly, we found that undifferentiated spermatogonia contained strongly H4K20me2 and H4K20me3, but little H4K20me1, whereas the differentiated spermatogonia possessed H4K20me1 and H4K20me2 and little H4K20me3. The findings of this study help for the understanding of epigenetic modifications during spermatogenesis in pigs and provide information for further studies.

324 TESTIS-SPECIFIC EXPRESSION OF Oct4-EGFP TRANSGENE IN PIG

Oct4 is a transcription factor essential for establishment and maintenance of pluripotency in mammalian stem cells. Oct4 expression was found in early embryos and germ cells throughout fetal development. In male mice, Oct4 expression is found in mitotically arrested prospermatogonia until birth. After onset of spermatogenesis, expression is maintained in type A spermatogonia, but is downregulated in type B spermatogonia and in spermatocytes (Pesce et al. 1998 Mech. Dev). Previously, we successfully generated Oct4-EGFP reporter pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev.). This animal model is unique because it allows in vivo and in vitro visualisation of Oct4-positive cells. Germ line specific Oct4-EGFP expression was analysed in testis isolated from young (&lt;1 week) and adult (&gt;7 months) pigs. Squash preparation of testicular tissue isolated from adult transgenic boars revealed high amounts of EGFP-positive cells compared to young piglets. We confirmed Oct4 and EGFP expression in the testis from young and adult transgenic animals using Northern blot analysis. Specific expression of Oct4 and EGFP in testis could be observed in blots as a single band of 1.5 kb. As a loading control, the blot was rehybridized with a β-actin probe. Mammalian testes contain different cell types, including germ cells, Sertoli cells, Leydig cells, and peritubular cells. To define the cellular origin of EGFP-expressing cells, we isolated these cells from adult transgenic testis using fluorescence-activated cell sorting (FACS)-based techniques. Analysis of isolated EGFP positive cells with qRT-PCR demonstrated the presence of marker genes specific for undifferentiated (Oct4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4, and GFRα1) and differentiated (BOLL and PRM2) germ cells. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of EGFP-positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the EGFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous Oct4 expression profile, Western blot and histochemical analyses are currently underway.

Altered Expression of Porcine Piwi Genes and piRNA during Development

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2–fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes.

Are expression and localization of tight and adherens junction proteins in testes of adult boar affected by foetal and neonatal exposure to flutamide?

Several recent studies have indicated that androgen disruption induced by the administration of anti-androgen flutamide during critical developmental stages results in various reproductive abnormalities, mainly in rodents. However, scarce data are available regarding the alterations caused by this toxicant on cell-cell adhesion molecules. Of note, there is no report on the expression and regulation of tight and adherens junction proteins in the boar. Therefore, the purpose of this study was to analyse whether foetal and neonatal exposure to flutamide affects the expression and distribution of ZO-1, occludin, β-catenin, and N-cadherin in testes of adult pigs. Moreover, to evaluate whether androgen signal was altered in the boar, testicular levels of testosterone and oestradiol and the expression of androgen receptor were examined. Flutamide (50 mg/kg bw) was injected into pregnant gilts during gestational days 20-28 and 80-88 (GD20, GD80), and into male piglets on postnatal days 2-10 (PD2). In the testes of all flutamide-exposed boars, expressions of ZO-1, N-cadherin and β-catenin were significantly decreased at mRNA and protein level, whereas expression of occludin was unchanged when compared with the controls. In addition, in severely damaged seminiferous tubules of PD2 pigs, mislocalization of ZO-1, N-cadherin and β-catenin was observed. Changes in junction protein expressions were accompanied by disturbed intratesticular androgen-oestrogen balance, although androgen receptor expression was not altered. Taken together, these results demonstrate that blockade of androgen action by flutamide during both gestational and neonatal periods affects the expression of ZO-1, N-cadherin and β-catenin in adult pig testes. Of concern, neonatal window seems to be most critical for the organization of BTB and consequently for normal spermatogenesis in the boar. It is likely that altered expression of junction proteins is related to insufficient testosterone production and/or excessive oestradiol synthesis, which may result from impaired Leydig cell function.

Fish oil diets alter the phospholipid balance, fatty acid composition, and steroid hormone concentrations in testes of adult pigs

The objective was to determine the effect of long-term dietary supplementation of two types of fish oil on lipid composition and steroidogenesis in adult pig testis. Twenty-four Duroc boars, aged 204.5 ± 9.4 d (body weight 128.1 ± 16.7 kg) received daily 2.5 kg of an iso-caloric basal diet supplemented with: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO) containing 16% of eicosapentaenoic acid (EPA) and 18% of docosahexaenoic acid (DHA); or 3) 60 g of tuna oil (TO) containing 7% of EPA and 33% of DHA. After these diets were consumed for 7 mo, testicular hormones, phospholipid content, and fatty acid composition of individual phospholipids in testis were determined. Body and reproductive organ weights were not significantly affected by dietary treatments. Testicular tissue from boars fed a TO diet, followed by those receiving MO and AF diets, had the lowest level of phosphatidylethanolamine (TO < MO < AF; P < 0.01) but the highest sphingomyelin (TO > MO > AF; P < 0.01). For each phospholipid, boars fed either the MO or TO diet had increased total omega-3 fatty acids, particularly DHA (P < 0.01), by reciprocal replacement of total omega-6 fatty acids (20:4n-6, 22:5n-6). The MO diet increased EPA more than the other diets. Testicular concentrations of testosterone and estradiol were lower in boars fed a TO diet than a MO diet (P < 0.02). In conclusion, long-term dietary supplementation of fish oil, regardless of the EPA/DHA ratio, modified the fatty acid compositions in testis and affected steroid production of healthy adult boars, which may represent a promising models for future studies on fertility.

Differential Expression of Connexin 43 in Adult Pig Testes During Normal Spermatogenic Cycle and After Flutamide Treatment

Evidence is mounting that the foetal and neonatal period of reproductive tract development is highly sensitive to hormonal disruption induced by various endocrine active compounds. Thus, we asked whether androgen withdrawal caused by prenatal (GD20, GD80) or neonatal (PD2) exposure to an anti-androgen flutamide alters Cx43 gene expression and may induce delayed effects on morphology and function of adult pig testes. Flutamide was given in five doses (50 mg/kg bw). Our histological analysis and TUNEL staining revealed varying degrees of seminiferous tubules abnormalities in all experimental pigs. Testes of pigs exposed to flutamide in utero exhibited moderate alterations of the spermatogenic process, whereas those of exposed neonatally were severely impaired. The most striking effects were spermatogenic arrest, germ cell detachment and a statistically significant increase in the frequency of germ cell apoptosis (p<0.01). Moreover, all pigs exposed to flutamide displayed Leydig cell hyperplasia. Because the network of cell-cell communication provided by gap junction channels plays an essential role in the regulation and maintenance of spermatogenesis, the physiological significance of Cx43-based gap junctions with regards to the gonadal impairment was evaluated by analysis of its expression using immunohistochemical, Western blot and qRT-PCR approaches. Significantly, lower Cx43 expression was found when flutamide was administered neonatally, which has coincided with severe disruption of spermatogenesis. Our data suggest that neonatal exposure to flutamide induces long-term effects on the spermatogenic capacity of the pig testis through alterations of Cx43-mediated intercellular communication and permanent alteration of both Sertoli and Leydig cell functions.

Expression pattern of acetylated α‐tubulin in porcine spermatogonia

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated alpha-tubulin (Ac-alpha-Tu) is a component of long-lived, stable microtubules and deacetylation of alpha-tubulin enhances cell motility, we hypothesized that acetylation of alpha-tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac-alpha-Tu at different stages of testis development was characterized by immunohistochemistry for Ac-alpha-Tu and spermatogonia-specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac-alpha-Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac-alpha-Tu appeared polarized toward the basement membrane. In adult pig testes, Ac-alpha-Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL-), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac-alpha-Tu. Comparison with the expression pattern of beta-tubulin and tyrosinated alpha-tubulin confirmed that only Ac-alpha-Tu is specific to germ cells. The unique pattern of Ac-alpha-Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348-352, 2010. (c) 2009 Wiley-Liss, Inc.

Differential expression of estrogen receptors (ERα/ERβ) in testis of mature and immature pigs

High affinity estrogen receptors (ERs) mediate estrogen action in male reproductive tissues. The objective of the present study was the immunolocalization of estrogen receptor alpha and estrogen receptor beta in immature and mature testes of pig, a species in which the role of estrogens on gonadal function is scarcely known. Testes from 3 and 18 month-old pigs were investigated. Immunohistochemistry was performed on paraffin embedded-tissues using both mouse anti-human monoclonal IgG ERalpha and IgG ERbeta 1 isoform. Western blot analysis demonstrated antibody specificity. ERalpha staining was not observed in immature testes, but it was detected in spermatogonia, spermatocytes and in the most Leydig cells of mature testes. ERbeta immunoreactivity was observed in spermatogonia and Leydig cells of immature gonads, while it was clearly detected in spermatogonia and in spermatocytes of adult pig testes. The differential ERalpha/ERbeta expression in germ and somatic cells of the gonads suggest a role of estrogens in function and in development of pig testis.

A two-step purification of cytochrome P-450 from adult pig testis by pregnenolone affinity column chromatography.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P-450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A purification of 755x was achieved with a yield of 3.09%.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Two-Step Purification of Cytochrome P-450 from Adult Pig Testis by Isoelectric Focusing

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17a-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.

The Relation between Two Molecular Species of P-450 in Adult Testis and 17α-Hydroxylase and 17,20-Lyase Activities

Two P-450s from adult pig testis were purified to specific contents of 11.2 and 12.0 nmol P-450/mg protein and shown to have minimum molecular weights of 45,000 and 46,000, respectively. The absorption spectra were typical of P-450s. The P-450s were separated from the two fractions by CM-C50 Sephadex column chromatography. One P-450 (Mr = 45000) exhibited 17,20-lyase activity of 6.78 nmol of androstenedione/min/nmol P-450, on incubation with 17α-hydroxyprogesterone as a substrate. The other P-450 (Mr = 46,000) exhibited no 17,20-lyase activity. Both P-450s exhibited 17α-hydroxylase activity that amounted to 10 nmol of steroid products. Accordingly, the two molecular species of P-450 are thus markedly different in 17,20-lyase activity toward 17α-hydroxyprogesterone.