T. H. Wise

Specific Staining of Sertoli Cell Nuclei and Evaluation of Sertoli Cell Number and Proliferative Activity in Meishan and White Composite Boars During the Neonatal Period

Abstract The positive relationship between Sertoli cell number and testicular size emphasizes the importance of determining factors involved in the regulation of the Sertoli cell population. Based on data from other species and indirect evidence in the boar, it is generally accepted that porcine Sertoli cells proliferate rapidly throughout the early postnatal period. However, direct evaluation of Sertoli cell number and the proliferative activity of Sertoli cells during the early postnatal period in boars have not been reported. Stereological enumeration of Sertoli cells is a labor-intensive process and would be greatly facilitated by a marker for these cells especially in the sexually mature male. Thus, the first objective of this study was to determine if expression of the transcription factor GATA-4 is an effective marker for fetal, postnatal, and adult Sertoli cells to facilitate enumeration procedures. The second objective was to evaluate the proliferative activity and growth of the Sertoli cell population in neonatal White Composite and Meishan boars, known to differ in mature testis size and Sertoli cell number, to determine the importance of this developmental period for the adult Sertoli cell population. GATA-4 was abundantly expressed by Sertoli cells throughout fetal and prepubertal stages of development and specifically stained both type A and B Sertoli cell nuclei in the sexually mature boar. Immunoreactivity was never observed in the germ cells regardless of their stage of development, illustrating that GATA-4 is a useful marker for both developing and adult Sertoli cells in the boar. Testicular size did not differ between breeds on Day 1 postpartum, but by 14 days postpartum White Composite boars had significantly larger testes compared to Meishan boars (P < 0.001). Similarly, Sertoli cell number did not differ between breeds at 1 day postpartum; however, at 14 days postpartum White Composite boars had a significantly larger Sertoli cell population compared to Meishan boars (P < 0.05). Surprisingly, despite having more Sertoli cells than Meishan boars at 14 days postpartum, the proportion of actively proliferating Sertoli cells in the White Composite boars was almost 50% less than the Meishan boars. This result illustrates that rapid rates of Sertoli cell proliferation probably occurred prior to 14 days postpartum in the White Composite boars. Collectively, these results illustrate that the relationship between testicular size and Sertoli cell number is manifested very early in the postnatal period for these two breeds. The substantial difference in the size of the Sertoli cell population and their proliferative activity between Meishan and White Composite boars during the early postnatal period emphasizes the importance of this early period for the establishment of the Sertoli cell population and subsequent adult testicular size.

Porcine gene discovery by normalized cDNA-library sequencing and EST cluster assembly.

Genetic and environmental factors affect the efficiency of pork production by influencing gene expression during porcine reproduction, tissue development, and growth. The identification and functional analysis of gene products important to these processes would be greatly enhanced by the development of a database of expressed porcine gene sequence. Two normalized porcine cDNA libraries (MARC 1PIG and MARC 2PIG), derived respectively from embryonic and reproductive tissues, were constructed, sequenced, and analyzed. A total of 66,245 clones from these two libraries were 5?-end sequenced and deposited in GenBank. Cluster analysis revealed that within-library redundancy is low, and comparison of all porcine ESTs with the human database suggests that the sequences from these two libraries represent portions of a significant number of independent pig genes. A Porcine Gene Index (PGI), comprising 15,616 tentative consensus sequences and 31,466 singletons, includes all sequences in public repositories and has been developed to facilitate further comparative map development and characterization of porcine genes (http://www.tigr.org/tdb/ssgi/). The clones and sequences from these libraries provide a catalog of expressed porcine genes and a resource for development of high-density hybridization arrays for transcriptional profiling of porcine tissues. In addition, comparison of porcine ESTs with sequences from other species serves as a valuable resource for comparative map development. Both arrayed cDNA libraries are available for unrestricted public use.

Sertoli Cells in the Boar Testis: Changes During Development and Compensatory Hypertrophy after Hemicastration at Different Ages

Abstract Changes in Sertoli cell numbers and testicular structure during normal development and compensatory hypertrophy were assessed in crossbred Meishan × White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56, or 112 days or to remain as intact controls through 220 days. The first testes removed were compared to assess testicular development. At 220 days, testicular structure was evaluated in boars representing the 25% with the largest (Lg) testis and the 25% with the smallest (Sm) testis in each treatment group. The number of Sertoli cells per testis reached a maximum by Day 56 in Sm testis but not until Day 112 in Lg testis boars, indicating a longer duration of Sertoli cell proliferation in Lg testis boars. Unilateral castration of Lg testis boars on Days 1, 10, 56, and 112 caused the weight of the remaining testis to hypertrophy by 149%, 135%, 119%, and 120%, respectively, and total sperm production to increase to 127%, 128%, 97%, and 106%, respectively. However, Sertoli cell numbers changed little in hemicastrate boars. In Lg testis boars, compensatory hypertrophy primarily involved proliferation of Leydig cells and expansion of existing Sertoli cells with little increase in Sertoli cell numbers, but in Sm testis boars, it involved expansion of existing Leydig and Sertoli cells without increase in cell numbers. These results indicate that Lg and Sm testis boars display intriguing differences during both development and compensatory hypertrophy, and they identify a unique animal model for further studies of factors that program and control Sertoli cell proliferation.

Interrelationships of Porcine X and Y Chromosomes with Pituitary Gonadotropins and Testicular Size

Abstract Endocrine and testicular responses to unilateral castration on 1, 10, 56, or 112 days of age were characterized in 132 Chinese Meishan (MS) × White composite (WC) crossbred boars in which testicular size associates with a quantitative trait locus (QTL) on X chromosome. At 220 days of age, testicles of boars unilaterally castrated on Day 1 or 10 weighed more and had greater total daily sperm production (DSP) than one testicle of bilaterally intact boars (P < 0.05); compensation did not double these two responses. Boars with MS alleles at the X chromosome QTL had smaller testicles, darker colored parenchyma, and lower total DSP than boars with WC alleles (P < 0.05). The MS alleles engendered greater (P < 0.05) plasma FSH and LH during puberty than WC alleles. Plasma FSH increased (P < 0.05) within 48 h of unilateral castration on Days 1, 10, and 56. Subsequent increases occurred earlier during puberty (P < 0.05) after unilateral castration at younger ages than after unilateral castration at older ages. Pubertal increases in plasma FSH and LH were greater (P < 0.05) in boars with MS alleles than in those with WC alleles for the X chromosome QTL. Breed of Y chromosome had no effect on testicular traits, FSH, testosterone, or estrone. For LH, boars with an MS Y chromosome had greater (P < 0.01) plasma LH across all ages than boars with a WC Y chromosome. We conclude that a gene or groups of genes that reside on the porcine X chromosome regulate testicular development and pubertal gonadotropin concentrations.

Association analyses of candidate single nucleotide polymorphisms on reproductive traits in swine.

The ability to identify young females with superior reproduction would have a large economic impact on commercial swine production. Previous studies have discovered SNP associated with economically important traits such as litter size, growth rate, and feed intake. The objective of this study was to test for association of candidate SNP with sow prolificacy reproductive traits in gilts of a Landrace-Duroc-Yorkshire composite population. Association analyses regressed additive (A), dominant (D), and imprinting (I) SNP effects on each trait with an animal model. A carnitine palmitoyltransferase 1A SNP and a glycogen synthase 1 SNP were associated with age at puberty (AP; D = 10 d; P = 0. 0037 and A = 3.8 d; P = 0.0078, respectively). Four IGF2 SNP were associated with AP as well, having additive or dominant effects (3.2 to 5.8 d; P < or = 0.0052). Two mannosidase 2B2 SNP and 2 prolactin receptor (PRLR) SNP were also associated with AP. Solute carrier 22, subfamily member 5 SNP was weakly associated with AP (D = 3.9 d; P < 0.10). Polymorphisms within glycogen synthase 1 and protein kinase AMP-activated, gamma 3 noncatalytic subunit had associations with ovulation rate. Estrogen receptor (ESR) 1, ESR2, PPAR gamma coactivator 1, and IGFBP3 SNP were significantly associated with weaning-to-estrus interval. Two PRLR SNP were associated with total number of piglets born (A = 0.57 piglets; P = 0.0095 and D = 0.61 piglets; P = 0.0016, respectively). A SNP within PRLR was also associated with number of piglets born alive (D = 0.61; P = 0.0016). The PPAR gamma coactivator 1 SNP was associated with total number of piglets born (D = 0.38 piglets; P = 0.0391) and number of piglets born alive (D = 0.53 piglets; P = 0.0032). The SNP within ESR1 (A = 0.65 piglets; P = 0.0950), ESR2 (A = -0.33 piglets; P = 0.0176), IGF2 SNP (A = -0.26 piglets; P = 0.0032), and IGFBP3 SNP (D = 0.35 piglets; P = 0.0683) were associated with number of piglets born dead. A leptin SNP was associated with mummified fetuses (D = 0.09 piglets; P = 0.0978). Many of the SNP analyzed in this study are from genes involved in regulation of metabolism, suggesting that there is an important link between physiological events associated with reproduction and energy utilization. Furthermore, these production and growth trait SNP may serve to assist in selection of young females for superior reproductive performance.

A Variant of Porcine Thyroxine-Binding Globulin Has Reduced Affinity for Thyroxine and Is Associated with Testis Size

Abstract The field of genomics applies the dissection of genetic differences toward an understanding of the biology of complex traits. Quantitative trait loci (QTL) for testis size, plasma FSH in boars, and body composition (backfat) have been identified near the centromere on the X chromosome in a Meishan–White Composite resource population. Since thyroid function affects Sertoli cell development and adult testis size in rodents, and thyroxine-binding globulin (TBG) maps to this region on the porcine X chromosome, TBG was a positional candidate gene for testis size. We discovered a polymorphism in exon 2 of the porcine TBG gene that results in an amino acid change of the consensus histidine to an asparagine. This single nucleotide polymorphism (SNP) resides in the ligand-binding domain of the mature polypeptide, and the Meishan allele is the conserved allele found in human, bovine, sheep, and rodent TBG. Binding studies indicate altered binding characteristics of the allelic variants of TBG with the asparagine (White Composite) isoform having significantly greater affinity for thyroxine than the histidine (Meishan) isoform. Alternate alleles in boars from the resource population are also significantly associated with testis weight. Therefore, this polymorphism in TBG is a candidate for the causative variation affecting testis size in boars.

Genetic relationships of body composition, serum leptin, and age at puberty in gilts.

Leptin produced by adipocytes acts through leptin receptors in the hypothalamus to control appetite and food intake and thus communicates information about degree of fatness. It is thought that a degree of body fat is required for initiation of puberty and maintenance of reproductive function in mammals. The objective of this study was to determine whether polymorphisms in the leptin (LEP), leptin receptor (LEPR), paired box 5 (PAX5), aldo-keto reductase (AKR), and pro-opiomelanocortin (POMC) genes were associated with age, leptin concentration, backfat as an indicator of body condition, or BW at puberty in 3 lines of gilts and to characterize genetic relationships among these traits. The first 2 lines, born in 2001, were formed by crossing maternal White Cross (Yorkshire x Maternal Landrace) gilts to Duroc (n = 210) or (lean) Landrace (n = 207) boars. The remaining line (n = 507), born in 2002, was formed by crossing progeny of the Duroc- and Landrace-sired lines. At first estrus, age, BW (BWP), and backfat (BFP) at puberty were recorded and blood was collected for leptin assays. Nine SNP were detected in candidate genes/regions: 1 in LEP, 3 in LEPR, 1 in PAX5, 2 in AKR, and 2 in POMC. Animals were genotyped for each of the SNP; genotypes were validated using GenoProb. The association model included fixed effects of farrowing group, covariates of SNP genotypic probabilities (from GenoProb), and random additive polygenic effects to account for genetic similarities between animals not explained by SNP. Variance components for polygenic effects and error were estimated using MTDFREML. Leptin concentrations were logarithmically transformed for data analysis. All 4 traits were moderately to highly heritable (0.38 to 0.48). Age and leptin at puberty had a significant (P < 0.01) genetic correlation at -0.63 +/- 0.097, and the genetic correlation between BWP and age at puberty was 0.65 +/- 0.083 (P < 0.01). Significant additive associations (a; P < 0.05) were detected at PAX5 for age at puberty (a = 3.2 d) and for BFP (a = 0.61 mm). One SNP in LEPR was associated with leptin concentration (a = 0.31 log units; P < 0.05). The associations from PAX5 correspond to a QTL peak for age at puberty detected on SSC1. Although not necessarily the causative mutation, this result implies that a QTL that can decrease age at puberty without increasing BFP and BWP at puberty may exist in this region in commercial pigs.

Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits

BackgroundThe rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL) for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10) near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR) gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine.ResultsScreening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4), which mapped to 126–128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07) and possibly ovulation rate (p = 0.102). Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03) and another possibly associated with age at puberty (p = 0.09).ConclusionAKR1C genotypes were associated with nipple number as well as possible effects on age at puberty and ovulation rate. The estimated effects of AKR1C genotypes on these traits suggest that the SNPs are in incomplete linkage disequilibrium with the causal mutations that affect reproductive traits in swine. Further investigations are necessary to identify these mutations and understand how these AKR1C genes affect these important reproductive traits.The nucleotide sequence data reported have been submitted to GenBank and assigned accession numbers [GenBank:DQ474064–DQ474068, GenBank:DQ494488–DQ494490 and GenBank:DQ487182–DQ487184].

Sertoli cell differentiation in pubertal boars.

Meishan boars experience puberty at a younger age than crossbred boars in association with earlier expansion of seminiferous tubules and smaller postpubertal testicular size. The current study defined changes in expression, assessed by immunohistochemistry, of anti-Mullerian hormone (AMH) and CDKN1B, markers of Sertoli cell differentiation, in prepubertal Meishan and crossbred (BX) boars and related these changes with the pubertal expansion of seminiferous tubules. Expression of AMH in tubules of Meishan and BX boars increased (P < 0.002) from 7 to 28 d of age. Pubertal development was characterized by declining AMH expression (P < 0.001), followed by increasing (P < 0.001) expression of CDKN1B in Sertoli cell nuclei and subsequent expansion of tubules. These pubertal changes occurred at younger (P < 0.001) ages in Meishan than in BX boars. In testes of 90-d-old BX boars, expression of CDKN1B in Sertoli cell nuclei and tubular diameter increased (P < 0.001) from the mediastinum outwardly toward the tunica. Evaluation of the same tubules in adjacent sections established that expression of AMH decreased followed by expression of CDKN1B in Sertoli cell nuclei; both changes occurred before tubular diameter achieved 90 microm. In BX boars unilaterally castrated at 90 d of age, tubular diameter was inversely related to weight of the remaining testis at 10 mo (P < 0.05), supporting terminal differentiation of Sertoli cells in a subpopulation of these boars. These studies established temporal relationships of AMH, CDKN1B, and seminiferous tubule diameter in pubertal boars of 2 genetically diverse lines and determined that differentiation of Sertoli cells during pubertal development progresses as a gradient from the mediastinum outwardly toward the tunica.

The relationship of plasma urea nitrogen with growth traits and age at first estrus in gilts

Gilts that reach puberty at an earlier age with more backfat have greater lifetime productivity. Increased growth rates generally promote earlier age at first estrus; however, an association of age at first estrus with discrete measures of body fatness remains controversial. We tested the hypothesis that metabolic state as determined by concentrations of plasma urea nitrogen (PUN), which reflect lean tissue growth, were correlated with age at first estrus. Blood samples were collected from gilts (n = 337) at 102, 123, and 145 d of age during development. Concentrations of albumin, creatinine, glucose, and PUN were determined. Body weight and backfat thickness were determined at each time point. From 130 to 240 d of age, gilts were monitored for first pubertal estrus. Concentrations of creatinine increased whereas concentrations of glucose decreased with increasing age (P < 0.0001). Concentrations of albumin and PUN remained relatively stable throughout development. Average daily BW gain (r = 0.22) and change in backfat thickness (r = 0.29) had a positive phenotypic correlation (P < 0.0001) with PUN at 145 d of age. Concentrations of PUN at 102 and 123 d of age were not phenotypically correlated with pubertal age, but there was a moderately negative phenotypic correlation (r = -0.22; P < 0.0001) of PUN at 145 d of age with age at first estrus along with a negative genetic correlation (r = -0.42). The relationship of PUN with age at first estrus shifted from liner to quadratic with advancing age. These data demonstrate that near the age at which gilts are selected for entry into the breeding unit, those with greater PUN have increased BW and backfat thickness and display pubertal estrus earlier but that PUN does not account for additional variation in age at first estrus beyond growth rate or backfat. It is concluded that PUN can be used to select gilts with increased efficiency of nutrient use without negatively impacting pubertal development.