Donald D. Lunstra

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.

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.

Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene

The objective of this study was to evaluate the reproductive risk associated with exposure of adult male Fisher-344 rats to inhaled benzo(a)pyrene (BaP). Rats were assigned randomly to a treatment or control group. Treatment consisted of sub-chronic exposure of rats via inhalation to 75microgBaP/m(3), 4h daily for 60 days, while control animals were unexposed (UNC). Blood samples were collected immediately after the cessation of exposures (time 0) and subsequently at 24, 48, and 72h, to assess the effect of bioavailable BaP on plasma testosterone and luteinizing hormone (LH) concentrations. Rats were sacrificed after the last blood collection. Testes were harvested, weighed and prepared for histology and morphometric analysis, and cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa. BaP exposure reduced testis weight compared with UNC (mean+/-SE; 2.01+/-0.11 versus 3.04+/-0.16g; P<0.025), and caused significant reductions in the components of the steroidogenic and spermatogenic compartments of the testis. Progressive motility and mean density of stored spermatozoa were reduced (P<0.05). Plasma testosterone concentrations were decreased by two-thirds in BaP-exposed rats throughout the time periods studied compared with those of their UNC counterparts (P<0.05), concomitant with increased concentrations of LH in BaP-exposed rats (P<0.05). These data suggest that sub-chronic exposure to inhaled BaP contribute to reduced testicular and epididymal function in exposed rats.

Effects of sexual experience, season, and mating stimuli on endocrine concentrations in the adult ram ☆

Two behavioral trials were conducted to determine the endocrine response of cortisol (C), luteinizing hormone (LH), testosterone (T), prolactin (PRL), and growth hormone (GH) in adult rams during exposure to estrous ewes during the breeding and nonbreeding seasons. One-half of the rams in each season were sexually experienced (SE) and the remainder were sexually inexperienced (SI). All SE rams (100%) achieved at least one ejaculation, but only 33% (summer) and 67% (fall) SI rams achieved ejaculation. In the fall, mean C, T, and GH concentrations were elevated (P less than .001) compared to values measured in the summer, whereas LH and PRL levels were higher (P less than .01) in the summer. Overall levels of C, LH, T, and PRL were higher (P less than .05) in SE rams than in SI rams. Mean GH concentration was higher (P less than .10) in SI than in SE rams during restricted and complete access to estrous ewes. In general, LH, PRL, and GH responses were similar during restricted and complete access to females for both SE and SI rams. Cortisol levels were higher (P less than .06) during periods of mating and T levels were higher (P less than .001) during periods where activity was limited to courtship behavior (nasogenital investigation). Correlations of hormones to reproductive behaviors indicated that mounting and intromission were associated with elevations in C and PRL, whereas elevated LH and T tended to be associated with courtship behaviors. Correlations between GH and behaviors were inconsistent. However, there was an increased coincidence between time of female exposure and hormonal response that occurred in the fall; brief exposure to estrous ewes resulted in increases in concentrations of all hormones examined. The most consistent response was observed in sexually experienced rams during restricted access to females during the breeding season. These results provide new information on the effects of season and level of sexual experience upon hormonal and behavioral characteristics of the ram during mating activity.

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.

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.

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.