Assessment of pubertal development of boars derived from ultrasonographic determination of testicular diameter.

At the onset of puberty, seminiferous tubules rapidly increase in diameter, thereby occupying a greater proportion of the testis, resulting in a rapid increase in testicular size. The objective of the current studies was to evaluate ultrasonography for assessing testicular diameter, as a basis for ranking boars relative to their extent of pubertal development. In the initial study, prior to castration at 4, 5, 6, or 7 mo of age, testicular length and diameter were assessed by ultrasonography in 160 anesthetized boars. After castration, testes were weighed. Mean diameter of seminiferous tubules and percentage of the testis occupied by tubules were determined by histological evaluations of all testes. Testicular volume was calculated from length and diameter and was correlated with testicular weight (P < 0.001; r ≧ 0.78) within each of the four age groups. At 4 and 5 mo of age, testicular diameter correlated positively (P < 0.001) with diameter of seminiferous tubules; this relationship was not significant at older ages. In two subsequent studies, testicular diameter determined ultrasonographically in conscious boars was highly correlated (r > 0.8) when assessed twice on the same day, or when diameter of the right was compared with diameter of the left testis. Similarly, testicular diameter obtained initially at 92 d of age correlated positively (P < 0.001) with the diameter observed at older ages, but the magnitude of the relationship decreased as time between evaluations increased. These findings supported ultrasonographic determination of testicular diameter during early pubertal development, as a means to rank boars of similar chronological age for extent of pubertal development.

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.

Association of allelic variants of thyroid-binding globulin with puberty in boars and responses to hemicastration.

Hemicastration of males increases weight of remaining testis when conducted before Sertoli cells cease to proliferate. The current studies re-examined responses to hemicastration in one-quarter Meishan crossbred boars that differed for two alleles of thyroid-binding globulin (TBG). In the first experiment, boars at 25 days of age with either allele did not differ in degree of testicular development; however, at 56 days, boars with the C allele had more advanced testicular development than littermates with the A allele as evidenced by testes with seminiferous tubules of larger diameter (P<0.008) and greater weight (P<0.05). At 10 months of age, boars hemicastrated at 25 days had a similar number of Sertoli cells in their single testis compared with both testes of control boars. However, in boars hemicastrated at 56 days number of Sertoli cells was less than the total number of Sertoli cells in the bilaterally intact controls; this reduction was greater (P<0.05) in boars with the C allele than in those with the A allele. The second experiment confirmed earlier (P<0.05) pubertal development in boars with the C allele relative to littermates with the A allele based on larger tubular diameter and the greater proportion of tubules with a distinct lumen at 60 and 80 days of age. These studies establish that boars with the C allele for TBG attain puberty at a younger age than those with the A allele thereby linking rate of pubertal development of boars with TBG or with gene(s) on the X chromosome in close proximity of TBG.

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.

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.

Breed differences in clearance of porcine FSH in hypophysectomized rats.

Extracts of anterior pituitary (AP) glands were infused i.v. into hypophysectomized male rats followed by sequential sampling of blood for 120 min. Determination of follicle-stimulating hormone (FSH) concentrations established that FSH from Chinese Meishan males decreased in the circulation of rats more slowly than FSH in extracts of AP from crossbred occidental pigs (P<0.003). Additionally, FSH from AP extracts of castrated males disappeared somewhat more slowly (P<0.06) than FSH from extracts of boars. Evaluation of FSH by bioassay and radioimmunoassay yielded similar concentrations in AP from Meishan and crossbred boars. Serum testosterone concentrations increased with time through 90 min after infusion of AP, but the rate of increase of testosterone was not related to amount of luteinizing hormone (LH) that was administered indicating LH receptor saturation. Unexpectedly, the rate of increase in testosterone was more rapid with AP extracts from boars than with extracts from castrated males. Observations from the current study imply structural alterations of FSH in the AP of Meishan males relative to crossbred males allowing sustained concentrations in the circulation, and this FSH possesses similar activation of the FSH receptor. The amount of LH in the AP extracts saturated the LH receptors of the hypophysectomized male rats, but some factor in extracts of boars differed from those of castrated males.

Genetic variation in sperm production.

In boars, the primary determinant of daily sperm production is the number of Sertoli cells, which establishes testicular weight. The only breed comparison of foetal testicular development in boars contrasted two diverse breeds, White composite (WC, Landrace-Yorkshire) with Meishan, a Chinese breed that undergoes pubertal development at a young age and has small testicular size. During the prenatal period, the pattern of change in testicular development is similar in these two breeds with both having their greatest proportion of proliferating Sertoli cells at 90 days of gestation, and with WC boars possessing more Sertoli cells and greater mass of seminiferous tubules during the latter half of gestation. During the first month of life, Meishan boars accumulate Sertoli cells and mass of seminiferous tubules at a greater rate than WC boars, and Meishan boars undergo terminal differentiation of Sertoli cells at a younger age. Postpubertal boars, within each breed and crossbreds of the two breeds, with small testicular size have increased circulating concentrations of follicle-stimulating hormone. No direct breed comparisons of testicular development are apparent for postpubertal boars of other breeds. Accepting the limitations of data reported from different laboratories, Piau boars reach puberty at an older age and have a greater proportion of their testes occupied with seminiferous tubules than Meishan boars; both breeds have small testes. A gene or genes on the X chromosome code for small testicular size in Meishan crossbred boars; genetic determinants of testicular size and sperm production in other breeds remain to be identified.

Deciphering the pig genome to understand gamete production.

The field of livestock genomics has made considerable advances in the past decade. In the area of pig reproduction, a number of genome scans have identified several genomic regions associated with variation in reproductive measures ranging from ovulation rate, litter size and testis size. Additionally, several candidate genes have been associated with variation in litter size. These studies primarily focused on developing genetic markers to facilitate selection decisions. To date, their results have made minor contributions to commercial pig performance and our knowledge on the inheritance of complex phenotypes. With the availability of additional resources for pig, as well as from human and mouse studies, future studies should be directed to identifying genetic variation that affects biological processes. To reach this goal, teams of diversely trained scientists need to be formed that include geneticists, physiologists, molecular biologists and bioinformaticists. A diversified team of scientists equipped with all of the available research tools (genomic sequence data, expression arrays, knowledge of gene product functions, etc.) and appropriate swine populations should be able to decode the genome's hidden secrets on how it controls reproductive processes.

Lack of an association between plasma follicle-stimulating hormone concentrations and ovarian weight in prepubertal gilts.

Selection for increased number of corpora lutea in gilts is associated with increased plasma FSH concentrations during pubertal development. In the current study, 270 gilts from a control (CO) line and a line selected for increased ovulation rate (OR) were unilaterally ovariectomized at 85 d of age, and this ovarian weight was related to FSH concentrations at 65, 75, and 85 d of age. Gilts were produced during two farrowing seasons, spring and fall, and the age at first estrus was monitored from 160 to 250 d. Plasma FSH was greater in OR than in CO gilts at 65 (P < 0.01) and 75 d (difference in spring greater than in fall, P < 0.01), but FSH at these ages was not correlated with ovarian weight at 85 d. At 85 d, FSH did not differ in gilts of these lines; however, FSH was negatively correlated (r = -0.27, P < 0.01) with ovarian weight. The proportion of gilts detected in estrus was less for spring-born CO gilts than for spring-born OR or for fall-born CO and OR gilts (78 vs. 92%, season x line, P < 0.02). The age at first estrus was similar in the two lines but was earlier (P < 0.01) for spring-born than for fall-born gilts (194 vs. 204 d). Concentrations of FSH at each of the ages examined were not correlated with the age at first estrus. These observations support the conclusion that selection for a greater number of corpora lutea produces a correlated increase in plasma FSH during early pubertal development. This increase in FSH most likely reflects differences in FSH synthesis and release and not differences in the stage of pubertal development.

Stereological evaluation of Sertoli cell ontogeny during fetal and neonatal life in two diverse breeds of swine.

Chinese Meishan (MS) boars have smaller testes due to fewer Sertoli cells compared with White Composite (WC) boars. The objective was to describe Sertoli cell development relative to circulating FSH concentrations in fetal and neonatal MS and WC boars. Testes and blood samples were collected on days 60, 75, 90 and 105 postcoitum (dpc) and 1, 7, 14 and 25 postpartum (dpp). One testis was immunostained for GATA4 or Ki67 antigen to evaluate total and proliferating Sertoli cell numbers respectively. Testicular size was greater (P<0.01) in WC than MS boars at all ages, associated with a greater mass of interstitial tIssue. Tubular mass (P<0.01) was greater in prenatal WC boars, but postnatally increased more rapidly (P<0.001) in MS boars, exceeding WC boars by 25 dpp. Sertoli cell numbers increased with age, was greater (P<0.001) in WC than MS boars during prenatal development but increased rapidly (P<0.01) by 1 dpp in MS and thereafter was similar in both breeds. The proportion of Ki67-positive Sertoli cells was maximal at 90 dpc, declining thereafter, did not differ between breeds through 7 dpp, but was greater (P<0.05) in WC than MS boars at 14 and 25 dpp. Plasma FSH concentrations were greater (P<0.05) in WC than MS boars at 75 dpc. FSH concentrations were elevated at 105 dpc (MS) and 1 dpp (WC) but declined thereafter with advancing postnatal age in both breeds. This study illustrates that late gestation represents the period of maximal Sertoli cell proliferation. Despite asynchronous Sertoli cell population growth between breeds during early postnatal life, differential mature Sertoli cell numbers and testicular size are probably due to differences in duration of the proliferative period after 25 dpp, potentially regulated by Sertoli cell maturation and blood-testis barrier formation. These events were not associated with fetal or early postnatal changes in FSH secretion.

Endocrine and molecular influences on testicular development in Meishan and White Composite boars.

The aim of this study was to evaluate developmental changes in thyroid hormone and other key endocrine hormones/molecular markers produced by testicular cells, in relation to breed differences in proliferation and maturation of Sertoli cells and general testicular morphological development in Meishan (MS) and White Composite (WC) boars. Blood samples and testes were collected on days 60, 75, 90 and 105 post coitum (dpc) and days 1, 7, 14 and 25 post partum (dpp). Testes were immunostained for thyroid hormone receptor-beta1 (THRbeta1), GATA4, Müllerian-inhibiting substance (MIS), 17-alpha-hydroxylase (P450(c17)) and inhibin subunits (alpha, betaA, betaB). In addition, protein levels were determined by densitometry. Plasma concentrations of free triiodothyronine (T(3)) were greater in MS (hyperthyroid) compared with WC (hypothyroid) boars (P<0.01) during fetal life, but the reverse was evident postnatally. Elevated levels of free T(3) during fetal life were associated with increased levels of THRbeta1, suggesting increased thyroid responsiveness of the testis during this time, contrasting with observations during early postnatal life. Localization patterns of THRbeta1, MIS, GATA4 and the inhibin subunits were consistent with previous studies. MIS protein levels declined more rapidly (P<0.001) in MS compared with WC Sertoli cells postnatally, consistent with earlier maturation of Sertoli cells as indicated by our previous study. In this study, transient neonatal hyperthyroidism in MS boars during late gestation was associated with a decline in proliferation and early maturation of Sertoli cells, followed by early onset of puberty in this breed. These observations indicate a possible role for thyroid hormone in the modification of Sertoli cell development, thereby influencing growth and differentiation of the testis in pigs.

Sertoli cells in the boar testis: changes during development and compensatory hypertrophy after hemicastration at different ages.

Changes in Sertoli cell numbers and testicular structure during normal development and compensatory hypertrophy were assessed in crossbred Meishan x 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.

Expression levels of Mullerian-inhibiting substance, GATA4 and 17alpha-hydroxylase/17,20-lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine.

Sexual differentiation and early embryonic/fetal gonad development is a tightly regulated process controlled by numerous endocrine and molecular signals. These signals ensure appropriate structural organization and subsequent development of gonads and accessory organs. Substantial differences exist in adult reproductive characteristics in Meishan (MS) and White Composite (WC) pig breeds. This study compared the timing of embryonic sexual differentiation in MS and WC pigs. Embryos/fetuses were evaluated on 26, 28, 30, 35, 40 and 50 days postcoitum (dpc). Gonadal differentiation was based on morphological criteria and on localization of GATA4, Mullerian-inhibiting substance (MIS) and 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450(c17)). The timing of testicular cord formation and functional differentiation of Sertoli and Leydig cells were similar between breeds. Levels of GATA4, MIS and P450(c17) proteins increased with advancing gestation, with greater levels of MIS and P450(c17) in testes of MS compared with WC embryos. Organization of ovarian medullary cords and formation of egg nests was evident at similar ages in both breeds; however, a greater number of MS compared with WC embryos exhibited signs of ovarian differentiation at 30 dpc. In summary, despite breed differences in MIS and P450(c17) levels in the testis, which may be related to Sertoli and Leydig cell function, the timing of testicular differentiation did not differ between breeds and is unlikely to impact reproductive performance in adult boars. In contrast, female MS embryos exhibited advanced ovarian differentiation compared with WC embryos which may be related to the earlier reproductive maturity observed in this breed.

An integrated comparative map of the porcine X chromosome.

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.

Interrelationships of porcine X and Y chromosomes with pituitary gonadotropins and testicular size.

Endocrine and testicular responses to unilateral castration on 1, 10, 56, or 112 days of age were characterized in 132 Chinese Meishan (MS) x 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.

Identification of genomic regions controlling plasma FSH concentrations in Meishan-White Composite boars.

The Chinese Meishan (ME) breed of pig is unique for many reproductive traits. Compared with Western breeds of swine, ME females reach puberty earlier, ovulate more ova per estrus, and have greater uterine capacity, while intact males (boars) have smaller testes and extremely elevated plasma levels of pituitary-derived glycoprotein hormones. In an effort to identify the genetic mechanisms controlling the elevated plasma levels of pituitary-derived glycoprotein hormones [in particular, follicle-stimulating hormone (FSH)] and to determine whether some of these genetic factors are also responsible for differences in other phenotypes, we scanned the entire genome for regions that affected plasma FSH in boars from a Meishan-White Composite (equal contributions of Chester White, Landrace, Large White, and Yorkshire) resource population. Initially, the entire genome of 121 boars was scanned for regions that potentially influenced plasma FSH. The most significant genomic regions were further studied in a total of 436 boars. Three genomic regions located on chromosomes 3, 10, and X apparently possess genes that significantly affect FSH level, and one region provided suggestive evidence for the presence of FSH-controlling genes located on chromosome 8. The region on the X chromosome also affected testes size. Similar genomic regions to those identified on chromosomes 3, 8, and 10 in this study have been identified to affect ovulation rate in female litter mates, supporting the hypothesis that plasma FSH in pubertal boars and ovulation rate in females is controlled by a similar set of genes.

Increased plasma follicle-stimulating hormone concentrations in prepubertal gilts from lines selected for increased number of corpora lutea.

Plasma follicle-stimulating hormone (FSH) was evaluated in gilts from two studies in which ovulation rate was increased through direct selection for number of corpora lutea (CL) to determine whether selection for ovulation rate affected FSH secretion during prepubertal development. In the first study, 76 control and 110 selected gilts of University of Nebraska gene pool lines were bled twice during prepubertal development. Plasma FSH concentrations were greater (P < 0.05) at 53 (13.5%) and 75 (21.3%) d of age in selected than in control gilts. In the second study, 254 control gilts, 261 gilts from a line selected for ovulation rate, and 256 gilts from a line selected for uterine capacity were bled at three prepubertal ages. Plasma FSH was greater (P < 0.05), relative to controls, on d 34 (> 24%), 55 (> 13%), and 85 (> 10%) in White Composite gilts selected for either increased ovulation rate or for greater uterine capacity. Unilateral ovariectomy and hysterectomy were performed at 160 d of age on random gilts in these three lines (n = 377); weights of these organs were evaluated to determine whether selection affected their development. Ovarian and uterine weights were less (P < 0.01) in the control than in the ovulation rate line. Subsequently, ovulation rate was determined during pregnancy (n > or = 130 gilts/line). Controls had fewer (P < 0.01) CL (14.6) than gilts of the ovulation rate line (17.7) but numbers similar (P > 0.10) to those of gilts of the uterine capacity line (14.7). Within each line, plasma FSH only on d 85 correlated positively with subsequent ovulation rate (P < 0.03, 0.001, and 0.08; r = 0.17, 0.30, and 0.15 for control, ovulation rate, and uterine capacity lines, respectively). Ovarian weight at 160 d of age also correlated with subsequent ovulation rate (P < 0.03 and 0.001; r = 0.23 and 0.38) in control and ovulation rate gilts but not in uterine capacity gilts (P > 0.10; r = 0.11). Gilts selected for increased number of CL, in two independent studies, had greater concentrations of FSH during prepubertal development than respective controls. The modest but significant, positive association of FSH at 85 d of age with subsequent ovulation rate provides additional support for using plasma FSH in prepubertal gilts to indirectly select for ovulation rate.

Temporal and spatial localization patterns of Gata4 during porcine gonadogenesis.

The zinc finger transcription factor Gata4, is associated with gonadal development in many species. The present study characterizes temporal and spatial localization of Gata4 throughout gonadogenesis in porcine embryos. Immunohistochemical studies illustrated that Gata4 protein is present in the coelomic epithelium prior to histological differentiation of the nascent bipotential gonad, marking the future site of both XX and XY porcine gonads. Many somatic cells of both XX and XY bipotential gonads continue to retain Gata4 immunoreactivity throughout sexual differentiation and subsequent gonadal development. Testicular cords were evident by 26 days postcoitum. Gata4 was present in Sertoli cells, identified by virtue of coexpression with Müllerian inhibiting substance and also interstitial cells including Leydig cells throughout fetal and postnatal life. Many somatic cells of the differentiating ovary including follicular cells also contained Gata4 protein throughout fetal and postnatal life. Gata4 was not present in germ cells, endothelial cells, or other undifferentiated mesenchymal cells of both XX and XY gonads. A population of Gata4-positive cells in the dorsal mesentery was continuous with the coelomic epithelium of the gonad. This localization pattern led to the hypothesis that a subpopulation of somatic cells in the dorsal mesentery moves toward the gonad. An in vitro cell migration assay demonstrated that Gata4-positive cells preferentially migrate toward explanted gonadal tissue, and morphological features of the developing gonad supported this hypothesis. This study illustrates that Gata4 is a very early marker for gonad formation, highlights species differences in temporal and spatial localization patterns, and suggests a potential role for Gata4 in the development of both XX and XY porcine gonads. Further, we suggest that mesenchymal cells of the dorsal mesentery may provide a source of somatic cells that migrate and incorporate into the gonad and contribute to various somatic cell lineages. Overall, the spatial and temporal localization patterns of Gata4 during porcine gonadogenesis implies a much earlier and wider role for Gata4 than previously reported in other species.

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.

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.