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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006510
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BIOLOGY OF REPRODUCTION 68, 140–150 (2003)
DOI: 10.1095/biolreprod.102.006510
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Sertoli Cells in the Boar Testis: Changes During Development and Compensatory Hypertrophy after Hemicastration at Different Ages1

D.D. Lunstra2,a, T.H. Wisea, and J.J. Forda

a USDA, ARS, R.L. Hruska U.S. Meat Animal Research Center, Reproduction Research Unit, Clay Center, Nebraska 68933-0166


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Leydig cells, male reproductive tract, puberty, spermatid, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is generally accepted that adult testis size is correlated with capacity to produce sperm and that total Sertoli cell numbers determine mature testis size in males of various mammalian species [1–6]. It is also accepted that Sertoli cell proliferation occurs prepubertally and that total Sertoli cell numbers are established both prior to formation of the blood-testis barrier and prior to puberty [6–11]. However, in swine, only two studies to our knowledge have enumerated Sertoli cells in mature [12] and neonatal boars [13] of conventional breeds, and no definitive studies have assessed changes in Sertoli cell numbers during postnatal testicular development in conventional boars. In Brazilian Piau boars, an exotic breed, one report states that Sertoli cell numbers increase dramatically between 1 and 4 mo of age [14]. In contrast, another report states that Sertoli cell numbers per testis decline by 40% between 3 and 5 mo of age in conventional boars [15]. Thus, enumeration and proliferation of porcine Sertoli cells during postnatal development remains to be clarified.

During prepubertal development in most species, normal testicular growth is associated with dramatic proliferation of Leydig cells in the interstitium and an increased number of Sertoli and germ cells within the seminiferous epithelium [5, 11, 13, 16–19]. Unilateral castration of prepubertal male mammals causes compensatory hypertrophy and increased size in the remaining testis, but hypertrophy generally occurs only if unilateral castration is performed in prepubertal males [20, 21]. In most species, this hypertrophy has been associated with increased diameter and length of the seminiferous tubules [22–25], increased numbers of germ and Sertoli cells [8, 21, 26–29], and increased sperm production per testis at maturity [22, 30]. Thus, the proliferative response to prepubertal unilateral castration is often used as a model for studying factors influencing testicular development. However, the few studies available regarding unilateral castration of boars have reported increased testis size [31, 32], some increase in Sertoli cell numbers after only short-term unilateral castration [15], or implied increases in Sertoli cell numbers inferred from increased mass of seminiferous tubules [25]. In prepubertal boars, definitive studies regarding the effects of unilateral castration on compartmental hypertrophy, Leydig cell numbers, and Sertoli cell numbers remain to be conducted.

Chinese Meishan (MS) boars have dramatically elevated serum gonadotropin and steroid concentrations throughout development, but they achieve markedly smaller adult testis size (paired testes, 300 g) and sperm numbers per ejaculate than mature boars (paired testes, 600 g) of conventional breeds in the U.S. swine industry [33–35]. In addition, a negative correlation exists between adult testis size and serum FSH concentrations in MS, conventional breeds and crossbred (MS x conventional) boars [36, 37]. Meishan boars reach puberty and produce sperm at a much younger age (70 days) and smaller pubertal paired testes size (40 g) than boars of typical U.S. breeds (120 days and 180 g, respectively) [35]. Although sperm production and fertility are maintained in the MS, aspects of testicular structure and functional efficiency are modified by accelerated reproductive development and continuous, long-term exposure to elevated hormone levels in MS boars [35, 37]. Because crossbred MS x conventional boars exhibit an extremely large range in adult testis size [36, 37], they provide a unique model for study of Sertoli cell proliferation and compensatory testicular hypertrophy.

Difficulties in visualizing the mammalian Sertoli cell by light microscopy have hampered structural elucidation and enumeration of this cell in the testis [6]. The primary feature of the Sertoli cell in light-microscopic preparations has been its pale-staining nucleus, which itself is not readily recognized and is difficult to identify, because the Sertoli cell nucleus is normally situated among more numerous and prominent types of germ cell nuclei [6, 21, 25]. Size of the mature Sertoli cell in mammals ranges from 2000 to 7000 µm3 (without the volume of embedded germ cells), and size of the Sertoli cell nucleus is approximately 600 µm3, with a range from 250 to 850 µm3 [6]. In mammals, Sertoli cell nuclei are usually located close to the periphery of the seminiferous tubule, near the basal lamina of the tubule. The nucleus in functional Sertoli cells is generally elongated or ovoid in shape, and irregular nuclear clefts increase the nuclear volume by approximately 15%. The interior of the nucleus displays an indistinct, fine granular texture and typically displays one very distinct, tripartite nucleolus that contains the condensed centromeric regions of most, if not all, Sertoli cell chromosomes [6, 8]. Because of the difficulty in identifying and enumerating Sertoli cells, relatively limited information regarding its structure and numbers is available for testicular development in most mammalian species [6, 38], and only sparse information regarding Sertoli cell numbers is available for swine.

The first objective of the present study was to assess changes in Sertoli cell numbers and structural aspects of normal testicular growth during postnatal development in crossbred MS x conventional boars. The second objective was to assess the effects of unilateral castration at 1, 10, 56, or 112 days of age on compensatory testicular hypertrophy, testicular structure, and Sertoli cell numbers in crossbred MS x conventional boars. After collection of morphometric data via traditional microscopy procedures conducted on tissue sections embedded in plastic without specific staining, a GATA4-staining procedure specific for Sertoli cell nuclei of boars became available [13]. Thus, a third objective was to compare Sertoli cell enumeration obtained from traditional procedures with that obtained from the GATA4-staining procedure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Design and Assignment of Boars to Treatment Groups

To obtain boars with large variation in testis size, 132 crossbred (1/2 MS x 1/2 White Composite ([WC]) boars were produced. The procedures used to select dams and sires for breeding, the mating program used to produce piglets, and the standard management program have been previously described [39, 40]. The mating scheme maximized the potential for variation in testis size based on the observation that plasma FSH concentrations in boars are negatively correlated with testis size [36, 37]. At birth, boars were assigned within sire to one of five treatment groups (unilateral castration of the right testicle on Day 1, 10, 56, or 112 or intact controls through Day 220; n = 28, 25, 25, 17, and 37, respectively). The Day 112 group was underrepresented to insure that at least 25 boars were assigned to each of the other groups. All surgical procedures conformed with approved guidelines for humane treatment of animals [41]. All boars in the present study were born in July and reached 220 days of age in February. Morphometry was restricted to the 25% of boars with the largest (Lg) testis and the 25% with the smallest (Sm) testis within each treatment group at 220 days of age, because morphometric assessment of testes and Sertoli cell enumeration is labor intensive. Assignment at 220 days of age to the Lg and Sm testis subgroups dictated that the first testis removed from those boars on Day 1, 10, 56, or 112 also be evaluated for developmental testicular structure and daily sperm production at those respective ages.

Collection and Processing of Tissues

Immediately after castration, testes and epididymides were trimmed and weighed. Each testis was cut in half longitudinally with a sterile scalpel, and one sample (6 x 6 x 6 mm; weight, ~0.2 g) of parenchymal tissue from the central portion was excised for immediate fixation and was then embedded in plastic for structural evaluation and Sertoli cell enumeration. For testes from Days 1, 10, and 56, the remainder of the parenchyma was snap-frozen for subsequent homogenization to determine daily sperm production per gram of testis (DSP; 4.37 day was used as DSP constant) and total daily sperm production per testis (TDSP). For testis from Days 112 and 220, approximately 10 g of the remaining parenchyma were snap-frozen for subsequent DSP evaluation; homogenization and determination of DSP and TDSP were performed as previously described [42]. For testes obtained on Day 220, three additional subportions (10 x 10 x 10 mm; weight, ~1.0 g each) of parenchymal tissue from the central portion were excised for immediate fixation and were then embedded in paraffin for GATA4 staining and enumeration of Sertoli cells [13].

Morphometric Assessment of Testes

For embedding in plastic, tissues were fixed immediately by immersion in 3.0% glutaraldehyde (no. 1909; Polysciences, Warrington, PA) in 0.15 M Dulbecco PBS (pH 7.4) for 4 h with gentle agitation, divided into subportions (approximately 3 x 3 x 3 mm each), fixed in fresh 3.0% (v/v) glutaraldehyde fixative overnight at 4°C with gentle agitation, washed in PBS (twice for 1 h each), dehydrated through a graded series of ethanol (50%, 70%, 80%, 95%, 100%, and 100% v/v; 1 h each), cleared in 100% propylene oxide (twice for 1 h each; Sigma, St. Louis, MO), and embedded in Luft Araldite 502 resin mixture [43] as described by Lunstra et al. [44]. Tissues infiltrated with plastic resin were polymerized into blocks in a temperature-controlled oven [42].

For general testicular morphometry on plastic sections, serial sections (thickness, 1 µm) were cut with glass knives from each of three plastic blocks per testis, mounted on glass slides overnight at 60°C, and stained with 1% periodic acid, 1% toluidine blue, and 0.5% basic fuchsin solutions [42]. Sections were initially evaluated at 100x magnification using a Zeiss Axioplan-2 Photomicroscope (Thornwood, NY) equipped with planapochromat objectives for differential-interference contrast (DIC) microscopy coupled with a computerized morphometry planimetry system (Bioquant Nova 2000 Advanced Image Analysis; R&M Biometrics, Nashville, TN) to obtain tubule diameters and area percentages occupied by seminiferous tubules, tubule lumens, and interstitium. Images of cells and structures from an Optronics DEI-750 triple-CCD color camera (Goleta, CA) attached to the microscope were displayed on a high-resolution (1600 lines/inch) color video monitor and were traced using a computerized mouse. Interstitial areas were evaluated at 400x magnification to obtain Leydig cell size and area percentages occupied by Leydig cells, myoid cells, and vascular structures (lymphatic and blood vessels). At least 75 round tubule profiles per testis were measured to obtain tubule diameter, and approximately 300 Leydig cells per testis were measured to obtain cell size. For all morphometric calculations, specific gravity of the testicular tissue was assumed to be 1.0, and area percentages were assumed to be equal to the volume percentages [45]. To adjust for capping effects, Leydig cell nuclear volumes were corrected using the Abercrombie formula [46]. No correction for section thickness was employed, because the diameter of cells and structures measured exceeded the section thickness by a factor of 10 or more [45]. No correction factor for shrinkage was applied, both because shrinkage using this fixative and embedding procedure is negligible [47] and because the samples used for comparison were fixed and processed into plastic blocks using identical conditions. The volume percentage of seminiferous tubules was multiplied by the total parenchymal volume per testis to obtain the total tubular volume and was then divided by the average area per round tubule profile, using the formula for volume of a cylinder, to obtain the total length of seminiferous tubules [12].

Enumeration of Sertoli Cells via Light Microscopy

For enumeration of Sertoli cells on plastic sections, two serial sections (thickness, 10 µm) were cut with glass knives from each of the three plastic blocks per testis and mounted on glass slides overnight at 60°C. Tissues within the profiles of at least 12 round tubules were selected at random and evaluated on the unstained sections at 1000x magnification using the DIC microscope and computerized morphometry system described above. The area inside each tubule basement membrane was evaluated by focusing sequentially down through the section, and the outline of each Sertoli cell nucleus was traced when in focus at its maximum diameter [12, 47]. Only nuclei with a distinct nucleolus were classified as Sertoli cell nuclei [6, 12, 47]. The area, perimeter, shape factor, longest diameter, and shortest diameter were recorded for each nuclear profile. Approximately 70 Sertoli cell nuclei per testis were measured, and the average volume per nucleus was calculated using the formula for a prolate sphere (4/3 {pi}ab2, where a = longest radius and b = shortest radius). The number of Sertoli cells per testis was calculated from the product of the volume percentage of Sertoli cell nuclei and the total parenchymal volume [47]. The volume percentage of Sertoli cell nuclei was multiplied by the total tubular volume per testis, after subtracting the total volume of tubule lumens when present, to obtain the total volume of Sertoli cell nuclei and was then divided by the average volume of a single Sertoli cell nucleus to obtain the total number of Sertoli cells per testis [47]. An index of the average size per Sertoli cell (SCS index) was obtained by dividing the total tubular volume per testis, after subtracting the total volume of tubule lumens, by the total number of Sertoli cells. Thus, the SCS index is an estimate of Sertoli cell volume that necessarily included the volume of all associated germ cells and cells from various stages of spermatogenesis when present at each respective age.

Enumeration of Sertoli Cells via GATA4 Staining

For embedding in paraffin (220 days of age only), tissues were fixed immediately by immersion in 4.0% paraformaldehyde (no. P-6148; Sigma) in 0.15 M PBS for 4 h with gentle agitation and postfixed in fresh fixative overnight at 4°C with gentle agitation. Each sample was washed in PBS (twice at each percentage for 1 h each), dehydrated through graded ethanol (50%, 70%, 80%, 90%, and 100% v/v; twice for 1 h each), cleared in xylene (twice for 1 h each; Sigma), infiltrated with paraffin wax (60°C, four times for 1 h each), and embedded in paraffin wax. For enumeration of Sertoli cells on paraffin sections at 220 days of age, four serial sections (thickness, 10 µm) were cut with stainless-steel knives from each of the three paraffin blocks per testis. Sections were dried overnight onto glass slides at 37°C and stored at room temperature until stained. Sections were deparaffinized, rehydrated, and stained for GATA4 to specifically identify Sertoli cell nuclei [13]. The volume percentage of seminiferous tubules and the average tubule diameter were evaluated at 100x magnification on fields selected at random using the DIC microscope and computerized morphometry system described above. No adjustment for tissue shrinkage in paraffin sections was employed, because tubule diameter averaged across all boars differed very little between plastic-embedded tissues (269 ± 5 µm; mean ± SEM) and paraffin-embedded tissues (262 ± 3 µm) at 220 days of age. To enumerate Sertoli cells at 1000x magnification, tissues within the profiles of at least 10 round tubules selected at random were evaluated on each 10-µm paraffin section. The area inside each tubule basement membrane was evaluated by focusing sequentially down through the section, and the outline of each stained Sertoli cell nucleus was traced when in focus at its maximum diameter [12, 47]. Only nuclei with distinct GATA4 staining were classified as Sertoli cell nuclei. Approximately 120 Sertoli cell nuclei per testis were measured, and the average volume of a single Sertoli cell nucleus, the total number of Sertoli cells per testis, and the average SCS index per Sertoli cell were calculated as described above.

Statistical Analyses of Data

Data were analyzed using the general linear model procedure of SAS Version 6.12 [48]. For analysis of testicular development from birth through 220 days of age, the model included the fixed effects and interactions of sire, age, and testis size subgroup. For analysis of compensatory hypertrophy at 220 days of age, the model included the fixed effects and interactions of sire, treatment group, and testis size subgroup. Sire rather than litter was included in analyses, because boars had been assigned within sire to respective treatment groups. All testicular composition data were transformed (log10) prior to statistical evaluation to adjust for heterogeneity of variances. Data are reported as least-square means and SEMs. Differences between treatment means were tested for significance using the predicted differences (PDIFF) procedure [48]. Testis data were not adjusted for the small differences in body weight between Lg and Sm testis subgroups, because the subgroups did not differ (P > 0.80) in body weight at final castration (Day 220, n = 58, 97.8 ± 2.7 kg), both because boar body weight and testis weight are not genetically correlated [49] and because such adjustments do not accurately represent the biology of boars exhibiting divergence in testis size [42].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Testicular Changes During Development

Changes in testis weight Testis weight did not differ (P > 0.13) between Lg and Sm testis subgroups at younger ages (Days 1, 10, and 56), but testis weight became larger in Lg testis than in Sm testis boars at older ages (Table 1). Testis weight in Lg testis boars was 148% that of Sm testis boars by Day 112 (P < 0.05), and Lg testis boars had 3-fold larger testis (P < 0.001) than Sm testis boars by Day 220 (Table 1).


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  		TABLE 1. Testes size, total daily sperm production (x109 per testis), tubular diameter, and total tubular length per testis in control and unilaterally castrated crossbred boars (1/2 Meishan x 1/2 White Composite).a

Onset of sperm production Onset of sperm production (indicated by presence of homogenization-resistant spermatid nuclei) first appeared in three of six Sm testis boars and in one of six Lg testis boars at Day 56. The TDSP was greater in Sm testis boars at Day 56 (P < 0.05) but had become approximately 4-fold greater in Lg than in Sm testis boars by Days 112 and 220 (P < 0.01) (Table 1). Testis weight was strongly correlated with TDSP (P < 0.001) during development in both Lg and Sm testis subgroups, respectively (r = 0.95 and 0.73, respectively). Diameter of seminiferous tubules averaged approximately 50 µm at Day 1 and Day 10 in both Lg and Sm testis boars (Table 1). By Days 56 and 112, tubule diameter was greater (P < 0.001) in Sm than in Lg testis boars, but tubule diameter became greater (P < 0.001) in Lg testis boars at Day 220. Total tubular length increased approximately 15-fold between Days 1 and 56 (Table 1) in both Sm and Lg testis boars but only doubled between Days 56 and 112 in Sm testis boars while increasing almost 5-fold in Lg testis boars during the same period. Comparatively, tubular length became more than 3-fold greater (P < 0.01) in Lg than in Sm testis boars at Days 112 and 220 (Table 1).

Changes in volumes and total masses of testicular compartments Total mass of tubules per testis was similar between subgroups at early ages, became transiently greater in Sm testis boars by Day 56, and was substantially greater in Lg testis boars by 112 and 220 days of age (P < 0.001) (Table 2). The volume percentage of seminiferous tubules was similar in both boar subgroups through 56 days of age, but it became significantly greater in Lg than in Sm testis boars by 112 and 220 days of age (volume percentage data not shown; can be interpolated from data in Tables 1 and 2). At 220 days of age, the volume percentage of seminiferous tubules (75 ± 2%) was approximately 4-fold greater than the volume percentage of Leydig cells (19 ± 2%) in Lg testis boars, whereas the volume percentages of seminiferous tubules (47 ± 2%) and Leydig cells (43 ± 2%) were essentially equal in Sm testis boars. The total mass of Leydig cells also was similar between subgroups at early ages; however, it was greater in Sm testis boars at Days 56 and 112 (P < 0.001) and became greater in Lg testis boars by 220 days of age (P < 0.001) (Table 2). However, total Leydig mass remained relatively similar between Sm and Lg testis boars throughout development despite major differences in testis weight (Table 1) and total tubule mass (Table 2).


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  		TABLE 2. Total tubular mass, Leydig mass, Leydig cell numbers per testis, and Leydig cell size in the testes from control and unilaterally castrated crossbred boars (1/2 Meishan x 1/2 White Composite).a

Changes in Leydig cells Total number of Leydig cells per testis was lowest at Day 1 in both Lg and Sm testis boars and increased steadily in both subgroups to reach maximums at Day 112 (Table 2). Leydig cell size exhibited a transient peak at 10 days of age in both Lg and Sm testis boars (Table 2). Leydig cell size was consistently larger in Sm than in Lg testis boars on Days 10, 56, and 112 (Table 2), but this difference had disappeared by Day 220. Correlations between total number of Leydig cells per testis and testis weight were higher in Sm (r = 0.81) than in Lg testis boars (r = 0.55) during development.

Changes in Sertoli cells Sertoli cell numbers per testis did not become significantly greater in Lg than in Sm testis boars until Day 56 (Table 3). At 112 and 220 days of age, Lg testis boars contained approximately 3-fold more total Sertoli cells than Sm testis boars (Table 3). Differences in total Sertoli cell numbers at Days 56, 112, and 220 were quite similar to the relative differences observed in total tubular length at those ages (Table 1). Number of Sertoli cells per testis in Sm testis boars was maximal by Day 56 (P < 0.001) and did not increase thereafter. However, Sertoli cell numbers in Lg testis boars did not approach maximum numbers until Day 112 (Table 2). Number of Sertoli cells per gram of testis was highest at Day 1 in both Lg and Sm testis boars and declined throughout postnatal testicular development (cells per gram data not shown; can be interpolated from data in Tables 1 and 3). Sertoli cells per gram did not differ (P > 0.50) in Lg and Sm testis boars by Day 220. Correlations between total number of Sertoli cells per testis and testis weight (r = 0.51 and 0.32 in Lg and Sm testis subgroups, respectively) and total tubular length per testis (r = 0.69 and 0.60, respectively) were higher in Lg than in Sm testis boars throughout development.


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  		TABLE 3. Total Sertoli cell numbers per testis, nuclear size, cell size index, and total daily sperm production per Sertoli cell in testes from control and unilaterally castrated crossbred boars (1/2 Meishan x 1/2 White Composite).a

Average nuclear size of Sertoli cells increased steadily throughout development and was largest at Day 220 (Table 3). Sertoli cell nuclear size was very similar at each age within Lg and Sm testis boars, except Sertoli cell nuclei were larger (P < 0.02) in Sm testis boars at Day 10. The average SCS index for individual Sertoli cells (calculated to include mass of germ cells) was larger in Sm than in Lg testis boars on Days 10, 56, and 112, but the SCS index did not differ (P > 0.60) by Day 220 (Table 3). Also, TDSP per Sertoli cell was higher in Sm testis boars only at Day 56 (P < 0.02) and did not differ (P > 0.20) in Lg and Sm testis boars at other ages. Daily sperm production per Sertoli cell averaged 2.0–2.2 ± 0.3 sperm at Day 220 in both Lg and Sm testis boars (Table 3).

Influence of Unilateral Castration on Testicular Traits

Testis weight Based on testis weight at 220 days of age, unilateral castration on Days 1, 10, 56, and 112 caused the remaining testis of Lg testis boars to hypertrophy by 149%, 135%, 119%, and 120%, respectively (P < 0.01) (Table 1) compared to control boars, and similar degrees of testicular hypertrophy were observed in Sm testis boars. Testis weight in respective control and hemicastrate groups was approximately 3-fold larger (P < 0.001) in Lg testis boars than in Sm testis boars (Table 1), regardless of age at unilateral castration.

Sperm production per testis Sperm production per testis (i.e., TDSP) was 127%, 128%, 97%, and 106% in Lg testis boars hemicastrated on Days 1, 10, 56, and 112, respectively, compared to controls, and this increase was significant in boars hemicastrated on Days 1 and 10 (P < 0.01) (Table 1). The increase in TDSP following unilateral castration also was dramatic in Sm testis boars, but it was significant only in boars hemicastrated on Day 1 (189%, P < 0.001) (Table 1). Similar to differences in testis weight, Lg testis boars exhibited TDSP per testis that was 3- to 4-fold higher than that of Sm testis boars, regardless of age at unilateral castration. Testis weight and TDSP were more highly correlated in hemicastrate (r = 0.66 and 0.66 in Lg and Sm testis subgroups, respectively) than in control boars (r = 0.45 and 0.59, respectively).

Volumes and total masses of testicular compartments Diameter of seminiferous tubules was not affected (P > 0.80) in Lg testis boars subjected to unilateral castration, but it was increased by approximately 50–70 µm in Sm testis boars (P < 0.05) compared to controls at 220 days of age, regardless of age at unilateral castration (Table 1). Unilateral castration tended to increase total tubular length in both Lg and Sm testis boars, and this increase was significant in Lg testis boars when hemicastration was performed at younger ages (P < 0.01). Unilateral castration in Lg testis boars had little effect on the volume percentage of the testis occupied by seminiferous tubules or Leydig cells at 220 days of age (data not shown). However, total mass of tubules and total mass of Leydig cells were increased by hemicastration in Lg testis boars (Table 2). Thus, compensatory hypertrophy increased the volume of both of these compartments almost equally in Lg testis boars. In Sm testis boars, unilateral castration caused the volume percentage of the testis occupied by seminiferous tubules to increase from 47% in controls to 54–58% in hemicastrates and markedly increased the total mass of tubules (Table 2) compared to control boars.

Leydig cell numbers Unilateral castration in Lg testis boars on Days 1, 10, 56, and 112 caused the total number of Leydig cells per testis to increase by 168%, 163%, 145%, and 118%, respectively (Table 2). In addition, the total mass of Leydig cells exhibited a substantial increase (Table 2), but the relative volume per Leydig cell changed little, compared to control Lg testis boars at 220 days of age. The increase in Leydig cell numbers in Lg testis boars was more pronounced when hemicastration was performed at younger ages (Table 2). Significant Leydig cell proliferation was not apparent in Sm testis boars, but total mass of Leydig cells tended to increase (Table 2). In lieu of Leydig cell proliferation in Sm testis boars, average volume per Leydig cell was increased by unilateral castration in Sm testis boars at every castration age compared to that in Lg testis boars (Table 2). Thus, compensatory hypertrophy caused a substantial increase in total mass of seminiferous tubules in both Lg and Sm testis boars, but the net effect of compensatory hypertrophy in the interstitium appeared to be accomplished via Leydig cell proliferation in Lg testis boars and via Leydig cell hypertrophy in Sm testis boars (Table 2).

Sertoli cell numbers Among control boars evaluated at 220 days of age, testis weight and Sertoli cell numbers were highly correlated (r = 0.83, P < 0.001), confirming that Sertoli cell numbers are strongly related to mature testis size in the boar. Surprisingly, unilateral castration had little effect on total Sertoli cell numbers per testis (Table 3). At 220 days of age, Sertoli cell numbers in Lg testis boars hemicastrated on Days 1, 10, 56, or 112 were 99%, 104%, 97%, and 97% (P > 0.20), respectively, compared to control Lg testis boars. In Sm testis boars, no increase in Sertoli cell numbers occurred (Table 3), and total Sertoli cells per testis even tended to decrease in some subgroups. The average SCS index of Sertoli cell size was increased (P < 0.05) in most Lg and Sm testis hemicastrate boars (Table 3) compared to respective control boars, regardless of age at unilateral castration. Also, Sertoli cell nuclear size was increased (P < 0.05) in hemicastrate groups compared to control boars at 220 days of age, regardless of unilateral castration age (Table 3), although increases were not significant in all subgroups. In general, the SCS index tended to be larger in Sm testis than in Lg testis boars, and relative differences in SCS index were reflected in Sertoli cell nuclear size (Table 3). Thus, in both Lg and Sm testis boars, increases in the seminiferous tubule compartment associated with hemicastration, evidenced by increases in tubule diameter, total tubular length, total tubular mass, and TDSP, reflected an increase in SCS index rather than an increase in Sertoli cell numbers per testis.

Sertoli Cell Enumeration Using GATA4 Staining

Enumeration of Sertoli cells in paraffin sections using GATA4-staining yielded results (Fig. 1) that were similar to data obtained for enumeration of Sertoli cells on plastic sections via DIC microscopy. Sertoli cell numbers enumerated from GATA4-stained sections at 220 days of age in Lg testis boars hemicastrated on Days 1, 10, 56, or 112 were 116%, 115%, 98%, and 102%, respectively, compared to control Lg testis boars at 220 days of age. Total GATA4-stained Sertoli cell numbers in Lg testis boars hemicastrated at Day 1 or Day 10 were higher (P = 0.03) than in control Lg testis boars (Fig. 1). However, the net increase in GATA4-stained Sertoli cells was quite small (16% and 15% in Lg testis boars hemicastrated at 1 or 10 days of age, respectively) compared to controls, and no significant increase in GATA4-stained Sertoli cell numbers occurred with hemicastration in Sm testis boars. Thus, enumeration of Sertoli cells via GATA4 staining confirmed the limited Sertoli cell proliferation in Lg testis and lack of Sertoli cell proliferation in Sm testis boars that occurred during prepubertal compensatory hypertrophy in the pig.



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  		FIG. 1. Comparison of Sertoli cell numbers per testis enumerated using two procedures: via morphometry of paraffin sections after GATA-4 staining of Sertoli cell nuclei (GATA4) and via traditional morphometry of unstained plastic sections using DIC microscopy. Data were obtained from testis of Lg and Sm testis boars at 220 days of age after hemicastration at 1, 10, 56, or 112 days of age and from respective control boars. Percentage values given above GATA4 columns indicate the degree of Sertoli cell proliferation present in hemicastrate boars for each hemicastration age compared to control boars within respective Lg and Sm testis groups. Horizontal dotted lines emphasize the lack of Sertoli cell proliferation in Sm testis boars (lower dotted line) and the very limited Sertoli cell proliferation in Lg testis boars (upper dotted line) that occurred after hemicastration

Enumeration via GATA4 staining showed slightly less variation, evidenced by smaller SEMs (Fig. 1), than did enumeration from unstained plastic sections. Total number of Sertoli cells per testis enumerated via GATA4 staining on paraffin sections was 7–20% higher per boar subgroup (average = 13.8% higher, P < 0.05) than that obtained from unstained plastic sections (Fig. 1). In addition, Sertoli cell nuclear size measured via GATA4-staining was slightly smaller than that obtained from unstained plastic sections when averaged across all boars at 220 days of age (614 ± 14 and 660 ± 19 µm3, respectively; P < 0.05). These differences in total Sertoli cell numbers and average nuclear volume were not caused by tissue shrinkage, because the tubule diameters in paraffin- and plastic-embedded tissues did not differ when averaged across all boars at 220 days of age (262 ± 3 and 269 ± 5 µm, respectively; P > 0.20).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Changes in Testicular Characteristics During Development

Results from the present study indicate that dramatic structural differences appear during development and that these contribute to the major differences in Sertoli cell number, mature testis size, and sperm production that exist in Lg and Sm testis crossbred boars. Similar 15-fold increases in total tubular length occurred between Day 1 and Day 56 in both Sm and Lg testis boars. Tubular length increased much more slowly beyond Day 56 in both groups of boars, supporting the conclusion that seminiferous tubules undergo their greatest rate of increase in length during the first few weeks after birth [50]. After 56 days of age, the degree of increase in total tubular length was much greater in Lg (5-fold) than in Sm testis boars (2-fold), most likely reflecting a difference in duration of Sertoli cell proliferation. Moreover, total number of Sertoli cells per testis were similar in Lg and Sm testis boars through 10 days of age but became significantly larger in Lg testis than Sm testis boars at 56 days of age and beyond. Total Sertoli cell numbers in Sm testis boars reached a maximum by 56 days of age, whereas Sertoli cell numbers in Lg testis boars did not approach maximum numbers until 112 days of age. Thus, duration of Sertoli cell proliferation during the postnatal period appears to be the primary factor that determines differences between Lg and Sm testis boars in mature Sertoli cell number and subsequent mature testis size.

A higher proportion of Sm testis boars exhibited homogenization-resistant spermatids and had significantly larger tubular diameter, SCS index, and TDSP per Sertoli cell at 56 days of age than Lg testis boars. Collectively, these data indicate that Sm testis boars initiated spermatogenesis sooner and achieved onset of puberty earlier than Lg testis boars. Onset of spermatogenesis at 56 days of age in Sm testis and by 112 days of age in Lg testis boars agrees well with pubertal data reported by others for MS boars [35, 51] and conventional boars [25, 35, 52, 53], respectively. Establishment of the blood-testis barrier requires that tight junctions be established between adjacent Sertoli cells around the periphery of the seminiferous tubule, and onset of spermatogenesis cannot occur until after the blood-testis barrier has formed [6, 7, 9, 10, 21]. If Sertoli cells cannot proliferate after formation of the blood-testis barrier [5], then duration of Sertoli cell proliferation is longer in Lg than in Sm testis boars. Therefore, timing of factors that cause formation of the blood-testis barrier must differ between Lg and Sm testis boars. Whereas the identity of these factors remains unknown, our results justify further study of factors that end Sertoli cell proliferation, establish the blood-testis barrier, and dictate mature testis size.

Changes in Testicular Characteristics Because of Unilateral Castration

As expected, unilateral castration of boars on Day 1, 10, 56, or 112 caused the mass of the remaining testis to hypertrophy by 220 days of age in both Lg and Sm testis boars compared to control boars. Furthermore, both testicular compartments (tubules and interstitium) were increased almost equally by compensatory hypertrophy, but the degree of hypertrophy in both Lg and Sm testis boars was more pronounced when hemicastration was performed at younger ages. This agrees with the general compensatory hypertrophy effects reported to occur in boars [15, 25] and other species [21, 23, 24, 27, 29]. Putra and Blackshaw [15] reported that unilateral castration of boars at 2 mo of age resulted in only a small increase (11%) in total Sertoli cell numbers per testis 60 days later but that much larger increases in total Sertoli cell numbers occurred when boars were hemicastrated at 3, 4, or 5 mo of age. Their results are difficult to interpret, however, because they also reported that Sertoli cell numbers per testis in control boars declined by 40% between 3 and 5 mo of age. Kosco et al. [25] reported a doubling of parenchymal mass and seminiferous tubule length by 122 days of age in boars hemicastrated at 10 days of age, which they interpreted as an implied increase in the total number of Sertoli cells. These investigators did not evaluate boars long enough after puberty to assess effects on adult testis size, and they did not enumerate Sertoli cells.

GATA4 Enumeration of Sertoli Cells after Unilateral Castration

The similarity in Sertoli cell numbers enumerated using the GATA4-staining procedure and those obtained using traditional morphometric procedures serves to confirm the limited Sertoli cell proliferation that occurred in response to prepubertal hemicastration of boars. Within seminiferous tubules, GATA4 staining is specific for porcine Sertoli cell nuclei and markedly reduces the time required for accurate enumeration of Sertoli cells, especially in tubules displaying the myriad of germ cell types that are present during spermatogenesis [13]. Whereas the GATA4-staining procedure produced somewhat higher total Sertoli cell numbers and smaller Sertoli cell nuclear size than obtained with traditional techniques, relationships between treatment groups and interpretation of results remained the same. Staining with GATA4 identifies nuclei of typical Sertoli cells (type A) and smaller nuclei of type B Sertoli cells; the latter are more difficult to accurately identify in boar testes [6, 13, 54]. Thus, higher Sertoli cell numbers and smaller nuclear size obtained at 220 days of age with GATA4 staining reflect more accurate enumeration of all Sertoli cell nuclei, especially the smaller, type B Sertoli cell nuclei.

Effects of Unilateral Castration on Sertoli Cell Proliferation

It is widely accepted that Sertoli cell numbers in adult animals determine adult testis size and that Sertoli cell numbers can only be manipulated prepubertally and cannot change after establishment of the blood-testis barrier. However, several reports question the totality of this hypothesis. In many seasonally breeding species, seasonally regressed testes exhibit dramatic reductions in spermatogenesis and Sertoli cell size [38]. In adult red deer of northern Europe, testis size is large in autumn but 3-fold smaller in spring, and the number of Sertoli cells per testis is 1.4-fold larger in autumn [55]. In the adult equine, testis size is 131% larger, TDSP is 176% larger, and total Sertoli cells per testis is 154% greater in spring (May–July) than in winter (November–January) [56]. Prepubertal treatment of rats with exogenous FSH during the first 10 days of postnatal life increased Sertoli cell numbers per testis by 165% at 10 days of age but by only 118% at 90 days of age compared to controls [57]. Collectively, these reports indicate that the number of recognizable Sertoli cells can increase and decrease both before and after establishment of the blood-testis barrier. All boars in the present study were born in July and reached 220 days of age in February, but our study was not designed to assess the effects of season. Because others have reported that boars exhibit higher sperm production [58, 59], testis size [58], and serum testosterone [33, 58, 59] during fall and winter months, it is doubtful that season had detrimental effects on testis structure and Sertoli cell numbers of boars at 220 days of age in the present study.

In the present study, concomitant testicular hypertrophy and increased sperm production coupled with the very limited Sertoli cell proliferation in hemicastrate boars was similar to the effects that occur in rhesus monkeys when hemicastrated postpubertally [60]. In both boars and adult rhesus monkeys, normal spermatogenesis does not operate at maximum, because sperm production in the remaining testis increases during compensatory hypertrophy without an appreciable increase in total number of Sertoli cells. Interestingly, both pubertal boars and adult rhesus monkeys exhibit prolonged elevation of plasma FSH after hemicastration, which appears to mediate the increased sperm production via a stimulatory effect on differentiated spermatogonia [60]. In support of this concept, we have shown previously that adult MS boars exhibit elevated plasma FSH levels and higher sperm production per Sertoli cell than adult WC boars [35]. This increased sperm production occurs via reduced degeneration of differentiated spermatogonia during normal spermatogenesis [12, 35]. In the present study, both SCS index and TDSP per Sertoli cell were greater in Sm testis than in Lg testis boars hemicastrated at 56 and 112 days of age (Table 3). A quantitative trait locus (QTL) for pubertal FSH and testis size exists on the X chromosome of these MS crossbred boars [39, 40]. Retrospective genomic classification for this QTL indicated that of the 58 boars in the present study, 26 of 29 Lg testis boars carried WC alleles and 27 of 29 Sm testis boars carried MS alleles in this region of the X chromosome. Thus, a gene or group of genes on the X chromosome accounts for much of the difference between Lg and Sm testis boars. Plasma FSH levels in MS and WC QTL boars did not differ during the early postnatal period, and both levels exhibited a transient peak at 10 days of age. However, plasma FSH levels began to deviate thereafter and were 2- to 3-fold higher in MS QTL boars than in WC QTL boars from 56 through 220 days of age [40]. These findings indicate that differences in mature testis size occur without differences in early neonatal FSH concentrations in Lg and Sm testis boars [40]. However, the elevated FSH levels postcastration [40] probably contributed to the increased TDSP and higher sperm production per Sertoli cell that occurred in hemicastrate boars.

FSH Levels and Sertoli Cell Numbers in Other Species

Large differences in adult testis size that exist between normal mice and hypogonadal mice, which lack circulating gonadotropins, have been attributed to a postnatal reduction in Sertoli cell proliferation in the absence of gonadotropins [19]. Interestingly, Sertoli cells continue to proliferate in the testes of hypogonadal mice long after onset of puberty in normal mice (20 days of age), indicating that gonadotropins are required to stop Sertoli cell proliferation at the start of puberty [19]. Thus, the elevated levels of plasma gonadotropins present in Sm testis boars peripubertally [40] may accelerate onset of puberty and limit adult testis size via early cessation of Sertoli cell proliferation. However, this seems to be unlikely, because differences in Sertoli cell numbers were apparent at 56 days but differences in gonadotropin concentrations before Day 56 were very subtle [40]. Also in disagreement with this hypothesis, humans and mice expressing the fragile X syndrome exhibit increased prepubertal Sertoli cell proliferation and enlarged adult testes with normal structural features and spermatogenesis, but levels of FSH and FSH signal transduction in fragile X males do not differ from normal [61]. Furthermore, treatment of boars with FSH from 8 through 40 days of age caused no testicular hypertrophy by 100 days of age, but those investigators did not enumerate Sertoli cells and did not examine boars after puberty [62]. Collectively, these data suggest the biological system that controls proliferation of Sertoli cells in boars has both similarities and substantial differences from those functioning in rodents and other species.

Conclusion

In summary, this study indicates that differences in mature testis size and Sertoli cell number in Lg and Sm testis boars are not a consequence of different rates of Sertoli cell proliferation during prepubertal development. Instead, shorter duration of Sertoli cell proliferation in Sm testis boars relative to duration in Lg testis boars appears to be the primary factor contributing to differences in Sertoli cell number and adult testis size. In addition, the effects of prepubertal, unilateral castration were unexpected, because hemicastration caused substantial testicular hypertrophy and a concomitant increase in sperm production coupled with only very limited Sertoli cell proliferation in Lg and Sm testis boars. Compensatory hypertrophy in the boar increased the mass of the tubular compartment primarily via increases in existing Sertoli cell function (via increased Sertoli cell nuclear size, SCS index, number of germ cells, and TDSP per Sertoli cell) without appreciable increases in Sertoli cell numbers. This hypertrophy was more pronounced when hemicastration was performed at younger ages. These results imply that programming of the adult number of Sertoli cells in boars occurs prenatally and that this programming cannot be overridden by compensatory testicular hypertrophy induced via prepubertal unilateral castration.


   ACKNOWLEDGMENTS
 
The authors thank Alan Kruger, Sharidan Parr, and Susan Hassler for their skillful technical assistance; personnel of U.S. Meat Animal Research Center Swine Operations for care of animals; and Donna Griess for secretarial assistance. The authors are also grateful to S. McCoard for her advice and helpful suggestions regarding these studies.


   FOOTNOTES
 
1 Mention of trade names is necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the same by USDA implies no approval of the product to the exclusion of others that may also be suitable. Back

2 Correspondence: D.D. Lunstra, Reproduction Research Unit, P.O. Box 166, State Spur 18D West, Clay Center, NE 68933. FAX: 402 762 4382; lunstra{at}email.marc.usda.gov Back

Received: 19 April 2002.

First decision: 13 May 2002.

Accepted: 15 July 2002.


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 RESULTS
 DISCUSSION
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