Sertoli cell differentiation in pubertal boars1'2

J. J. Ford and T. H. Wise

USDA-AR.S, US Meat Animal Research Center. Cla y Center, NE 68933-0166

ABSTRACT: Meishan boars experience puberty ata younger age than crossbred boars in association withearlier expansion of seminiferous tubules and smallerpostpubertal testicular size. The current study definedchanges in expression. assessed mb iinunohistoclienus-try, of anti-Mullerian hormone (AMH) and CDKN1B,markers of Sertoli cell differentiation. in prepubertalMeishan and crossbred (BX) boars and related thesechanges with the pubertal expansion of seminiferoustubules. Expression of AMH in tubules of Meishan andBX boars increased (P < 0.002) from 7 to 28 cl of age.Pubertal development was characterized by decliningAMH expression (P < 0.001). followed by increasing(P < 0.001) expression of CDKN1B in Sertoli cell nu-clei and subsequent expansion of tubules. These pu-bertal changes occurred at younger (P < 0.001) agesin Meishan than in BX boars. In testes of 90-d-old BX

hoars. expression of CDKNIB in Sertoli cell nuclei andtubular diameter increased (P < 0.001) from the me-(liastinum outwardly toward the tunica. Evaluation ofthe same tubules in adjacent sections established thatexpression of AMH decreased followed b y expression ofCDKN lB in Sertoli cell nuclei: both changes occurredbefore tubular diameter achieved 90 pin. In BX boarsunilaterally castrated at 90 d of age. tubular diameterwas inversely related to weight of the remaining tes-tis at 10 nb (P < 0.05). supporting terunnal differ-entiation of Sertoli cells in a subpopulation of theseboars. These studies established temporal relationshipsof AMH. CDKN1B, and sennniferous tubule (lianleterat pubertal boars of 2 genetically diverse lines and de-termined that differentiation of Sertoli cells during pu-bertal development progresses as a gradient froni therrlediasti]iuIll outwardly toward the tunica.

Key words: anti-i\ lullerian hormone. CDKN1 B. porcine, puberty, Sertoli cell, testis

2009 American. Soc/ct!J of 4nuiial Scin.cc All rqhtsicscrved. .1. Anini. Sci. 2009. 87:253625J3(loi:1 0.2527/j as.2009- 1906

INTRODUCTION

Daily sperni production of adult males increases withnumber of Sertoli cells and testicular weight (Orth etal.. 1988; Huang and Johnson, 1996: Lunstra et al..2003). Sertoli cells undergo terminal differentiation andcease to proliferate during pubertal development coin-cident with formation of the blood-testis barrier (Gon-dos and Berndstou, 1993: Franca et al., 2005): however,in seasonal breeders, number of Sertoli cells decreasestliiriiig the nonbreeding season, but then increases atthe onset of the next breeding season (Hochereau-de

Mention of trade names or coninu-rcial products in this article issolely for the purpose of providing specific information and does notimply recommendation or endorsement by the USDA.

2We thank time following employees of the US Meat Animal Re-search Center: swine operations personnel for their dedicated servicefor care of animals and assistance with tissue collection: A. Kruger.Al. Ju(y. and P. Wood for superb technical support; and L. Parnellfor excellent assistance with manuscript preparation.

:'Corresponding author: joe.fordQars.usda.govReceived Februar y 19, 2009.Accepted April 23, 2009.

Reviers and Lincoln. 1978; Johnson et al., 1991: Tarulliet al., 2006). Recently, terminal differentiation of adultSertoli cells has been redefined as arrested proliferat-ing cells (Ahined et al., 2009). Less mature Sertohi cellssecrete abundant quantities of anti-Mullerian hormone(AIVIH) with AMH expression decreasing before pu-berty (Iran et al.. 1981: Rev et al.. 2003). In rodents.rapidly proliferating neonatal Sertohi cells express verylow CDKN1B. a cvclin-dependent kinase inhibitor alsoknown as 1)27"° 1 . whereas nonprohiferatmg Sertoli cellsin adult testes express increased levels of CDKNIB(Beumer et al.. 1999: Holsberger et al.. 2003). Timingof CDKN1B expression within Sertohi cells of pubertalboars and its relationslup with the pubertal decline inM\ IH remain unknown.

\ Eeishan boars are younger at puberty. have smalleradult testes, and produce less sperm than commercialcrossbred boars (Borg et al.. 1993: Lunstra et al.. 1997),thereby providing a valuable animal model to comparetemporal changes in AMH and CDKN lB relative toexpansion of seminif rous tubules. The second objec-tive of the current study was to define the pattern ofchange of these factors within testes and relate thesechanges to formation of the blood-testis barrier. Boars

2536

iit>Ii ((II I I illi.I(1iiIflhi)fl 2337

offer an advantage for such studies (tile to their moreprolonged rate of pubertal development relative to thewell-characterized, rapid changes in rodents (Sharp( , etA, 2003).

MATERIALS AND METHODS

Roars were born and reared at the US Meat AnimalResearch Center using standard product ion and ex-perimental practices that were in accordance vit Ii theGuide for the Care and Use of Agricultural Animals inAgricultural Research and Teaching (FASS, 1999).

Animals and Treatments

Boars were weaned at 17 to 20 d of age, reared illconfinement buildings, penned lw genetic line, and fedstandard diets formulated to me et NBC requirementsvarying with increasing age to accommodate changingmit cit ioiil need. Water was provided ad libitum. Thefirst experiment was conducted iii 2 parts. In the firstpart. 3 littermate boars from each of 4 litters of i\Ieis-han and a 4-breed composite maintained by inter semating (BX: Klindt et at. 2006) were randomly as-signed to castration at 7, 28. or 49 d of age. In thesecond part, 4 htterinate boars from 6 litters each ofJ\leishan and BX were assigned to castration at 49. 70,91. or 112 d of age.

In the second experiment. htterrnate boars front 20litters produced by 12 BX sires were assigned to unilat-eral castration at 7 ( ii = 20) or 90 (ii = 22) d of age.The remaining testis was removed at 10 mo of age.

Sample Collection and Processing

('astral ions at 7 d were conducted with a standardmanagement protocol without anesthesia. At 28 d.boars received lidocaine h ydrochloride (Western Veteri-nary Supply. Porterville. CA) as multiple, subcutaneousinjections iii the scrotal area 15 min before cast rat ion.On d 49 to 112 and at 10 mo. boars were tranquilizedwith xvlazine (2.3 mug/kg. Lin.: RX Veterinar y Prod-ucts. \Vcstlake. TX) 15 to 20 mimi before adminst er-ing thiopental sodium (Hospiara Inc.. Lake Forest, IL)intravenously, followed with closed-circuit anesthesiaof isoflurane (Halcarhon Laboratories, North Augusta,,SC) and oxygen.

After castration in Exp. 1, each testis was trinumned,weighed, and split longitudinally, and 2 aliquots of t is-sue. <1 cmiii , were placed in neutral buffered formalin forfixation. After approximately 5 h, tissues were placer]in fresh fixative for approximatel y 22 Ii more and thendehydrated with increasing concentrations of ethanoland inihedded in paraffin. In Exp. 2, testes obtained at90 ci of age were split longitudinally: 2 aliquots of tissuewere taken that contained a portion of the tunica to usefor later orientation. 1 piece from the upper one-halfamid 1 froni dic lower cue-half of the test is. Additional-

1, 2 pieces of tissue were taken approximately midwaybetween the tuiuiica and mediastm]nim. I from the lowerone-half and 1 from the upper one-half. Fixation andprocessin g were conducted as described above.

Imniunohistochemistr y was ('omldlmcted on S-tim sec-tions that were deparaffinized and exposed to AI\IHantibod y prepared in goats (SC-6886 for Exp. 1. Mc-Coard et al.. 2001. and SC-34833 for Exp. 2: SantaCruz Bioteclniologv, Santa Cruz. CA) or to CDKN1Bantibody prepared in rabbits (SC-528: Santa Cruz Bio-technology: Wang et al.. 2008). Availabilit y of a poly-clonal antibody against an internal region of AMH (SC-34833) permitted verification of observations with thefrequently utilized antibody against the C-terminus ofAMH (SC-6886). Nonimmune serum or absence of theprnna.m'anti antibodies was used to conclude that 11()ilspe-cific binding was not problematic. Additional negativecontrols included incubation of primar y antibodies withappropriate blocking peptides 1:1 overnmglit before use.SC-68861` and SC-528P (Santa Cruz Biotechnology).Blocking peptide was mmnt available for SC-31833. Theavuhmm-biotin inimnuimmoperoxidase s ystem was used to vi-sualize antibody binding (Vect ast air! Elite ABC Kit,Vector Laboratories Inc.. Burlingame, ('A). Novared(Vector Laboratories) was used as the chrouimogen. Tis-sue was visualized using light coumntersta illiug with he-matoxyhimi. All tissue sections within each experimentwere processed concurrently wit hi each antibody.

To quantify AMH, densitomnetric anal vses with a Bio-(hrmant Nova color-imaging system (Bioqimant Nova Ad-vanced Image Anal ysis 2000, II I\E Biometrics Inc..Nashville, TN) were used to determine the proportionof time pixels within randomly selected semimimuferons tu-bules that exceeded a threshold limit. Before each setof evaluatiomms, spectral density was standardized byfull-field optical histograms. For semi-quantification ofCDKN1B. the proportion of semnimñferous tubules withSertohi cell nuclei that stained definitivel y for CDKNIBwas determined. For Exp. I, >50 sennunferous tubules/block of tissue were evaluated for AMI-I. and >100 tu-bules/block were evaluated for CDKNI B .A similarnumber of tubules was selected at ramidomn from eachof the 4 quadrants of each tissue section. Fields of viewwere selected at random within each quadrant.

In Exp. 2, the tissue sections that were sampled nearthe tunica were evaluated as 2 regions, I regiomm be-ing <1.5 mum from the tumuica and the other definedas sub-timnica being >1.5 mum from the tunica. In timemuiediastinuni, sub-tummiica, amid tuimica regions. CDKN lBwas assessed in > 100 tubules per tissue block per boar.Tubules were selected randomly for evahimatiomi as de-scribed above. Additionally. the 16 boars with meandiameter of tubules near the mediastimmimmum of <100 p.m(range 68 to 100 mu) were stratified by meandiameterof semumimuiferonms tubules, and 6 that were distributedh)roportic)nally across this range were selected. The up-per limit for mean diamuiet cc was set- at 100 jitii becauseAu il-h u-as rarel y detected ivlmu'ii (liamet er ('\cu'e(l('dl this

2538 Ford and Wise

A

30-–°-Meishan

:

BX- -

All ages, P< 0.01

0 40 80 120

B

160 .

.

–o-Meishan **

120 BX

40 'Liiiege,P <0.02

0 40 80 120

C

300 –o-MeishanE BX/

/' 200-1

**E100

Line X Age, P<0.0 I0'

0 40 80 120Age, d

Figure 1. Chronological changes in B\V and testicular develop-inent in \leishan and crossbred (BX) boars. Body weight (A), pairtesticular weight (B), and mean diameter of seminiferous tubules (C)increased with age in both genetic lines of boors but at differing rates,5 P < 0.05 aial 001.

value. In serial sections of these 6 boars. identical tu-bules were identified in the tunica and sub-tunica re-gions (^: 10 tubules per tissue block/region for a total of262 tubules): 1 section was stained for AMH, and theadjacent section was stained for CDKN1B.

Mean diameter of seminiferous tubules was deter-mined from the circumference of nearly symmetricaltubules, >50 tubules per tissue block in Exp. 1 and, in

Exp. 2. >50 tubules per region per block of tissue foreach of the 3 regions. For each morphological trait, amean within hoar or a mean within region for each boarwas determined.

Statistical Analyses

Data from each part of Exp. 1 were evaluated sep-arately by Mixed Models procedures (SAS Inst. Inc..Cary, NC) with litter as a random effect (Littell etal., 1996). Body weight was evaluated separately foreach (lay of age. Data for other traits were evaluated

in a single anal ysis wit.luim each part. Data front these2 parts of this experiment were combined for presenta-tion. Mixed Models procedures with boar as a. blockand the 3 regions within each! testis compared with oneanother using the PDIFF option were used to evaluatedata front Exp. 2. Regression analyses were conductedwith I he Proc regression option of SAS. To evaluatethe affect of age at unilateral castration on the rangeof testicular weight at 10 mo of age, testicular weightclasses were compared with the Proc GENMOD optionof SAS adjusting for the influence of litter within sire.Data are presented as means + SEM.

RESULTS

Exp. 1 Breed Comparisons of SeminiferousTubule Development

Meishan boars were smaller (P < 0.01) at birth andgrew at a slower rate than BX boars (Figure 1A). Tes-ticular weight was similar in boars of both genetic lines

at 7. 28. and 49 cl (Figure 113), after which testicularweight increased more rapidly in Meishan than in BXboars (P < 0.02). Diameter of seminiferous tubules wassimilar at 7 and 28 4 of age (Figure 1C) but increasedmore rapidly with age in i\ leishan than in BX boars (P< 0.01).

In both genetic lines. AMH expression increased (P< 0.002) from 7 to 28 d (Figure 2A) and was greater (P< 0.001) in BX than in Meishan boars through 70 cl.Expression of AMH declined after 28 4 in both genet iclines but at a. more rapid rate (P < 0.001) in Meislianthan in BX boars and was nearly absent at 70 cl inMeishan and at 112 4 in BX boars. Within the semi-niferous tubules. AMH was localized in the cytoplasmof Sert.oli cells and was not detected iii germ cells. De-tection of CDKN1B in Sertohi cell nuclei became ap-parent at 28 4 in sonic Meisha.n and at 70 4 in someBX boars (Figure 213), after which the proportion oftubules containing Sertoli cells positive for CDKN I Bincreased rapidly (P < 0.001) in each line of boars butwas greater in Meishan than in BX boars from 49 to91 d (P < 0.003). Considerable variation in amount ofseiriirriferoims tubule development was apparent withinthe testes of a few 49-d-old Meislian and 91-d-old BXboars (Figure 3).

Exp. 2—Pattern and Significance of SertoliCell Differentiation

To establish whether seimnniferous tubule developmentis randoni or Patterned testes of 22 BX boars at 90 dof age were examined to assess the temporal relation-ship of CDKN113 expression iii Sertohi cell nuclei andexpansion of sennniferous tubules. Testicular weight, ofthese boars ranged from 12.7 to 33.6 g (rican = 22.4± 1.3 g). Mean diameter of senuniferous tubules was

greater in the ineclia,st.inum (91.8 ± 1.46 pm) comparedwith the tuniea (76.0 + 1.46 In n: P < 0.001) and sub-

l'UaIHI (LII dl jj Y) ('111 I ((((I

2539

60 A **

—o- Meishan

. . 40-**

**

20

Line X Age,

P<O.91

0 40 80 120

B

Meishan

),Age,*P3/

0 40 80 120Age, d

Figure 2. Changes at anti- Mullerian llorrnoral A \ TM) and CD-KNIB expression with age within the senuniterous tubules of Meishanand crossbred (MX) boars. These data were derived from 2 separatestudies. The first evaluated boors from i to 49 ci of age (4 boars/age,/genetic line). The second evaluated boors trout 49 to 112 d of age (6boors/age/genetic line). Separate statistical anal yses were conductedon data from each stud y. ** P < 0.01.

tunica region (86.2 ± 1.46 p.m: P < 0.01). Mean tubulardiameter in each of these regions correlated positively(P < 0.001) with each other and with weight of thetestis at 90 d of age: for diameter vs. testis weight (r2

= 0.77, 0.64. and 0.51 for the inediastinuin, sub-tunica.and tunica regions. respectively ). Percentage of tubuleswith Sertoli cell nuclei that, were positive for CDKN1Bwas greater near the mediastimim (63.4 + 2.2%) thanin the tuiiica (49.0 + 2.2 (X: P < 0.001) and the sub-tunica region (56.8 ± 2.2 (/c: P < 0.038). Percentage oftubules with Sertoh cell nuclei positive for CDKN1Bincreased (P < 0.003) concomitantl y within each of the3 regions and increased with diameter in the 3 regionsof the testes: for CDKN1B vs. diameter. 1- 2 = 059. 0.66.and 0.11 for the methiastiiiuni, sub-tunica., anti tunicaregions, respectively.

Evaluation of the same tubules in serial sections re-vealed that the intensit y of AMH decreased concurrentwith increasing tubular diameter (P < 0.001) near thetunica (Figure 4A) and in the sub-tunica region (Figure4B). Presence of CDKN1B within Sertoli cell nucleiincreased (P < 0.001) with increasing diameter of tu-bules. The majority of seminiferous tubules less than65 1dm in diameter were positive for AMI-1 and negativefor CDKN1B. In contrast, most tubules greater than 80un in diameter were positive for CDKN1B but nega-tive for AMH.

Evaluating testicular weight at 10 mu of age assessedthe impact of degree of Sert,oli cell differentiation at thetnne of unilateral castration. Boars that were unilater-ally castrated at 7 ci of age had heavier (P < 0.028)testicular weight than boars that were unilaterally cas-trated at 90 ci of age (634 + 22 vs. 566 + 21 g). Thisdifference resulted from a change in distribution of tes-ticular weight at 10 mo of age (P < 0.001) for these 2groups of boars (Figure 5): 23% of the boars unilater-ally castrated at 90 d had testicular weight less than450 g compared with no boars in this testicular weightclass for boars that were unilaterally castrated at 7d. Mean diameters of the seminiferous tubules in themediastinum (P < 0.043) and sub-tunica (P < 0.017)regions of the testis removed at 90 d of age were nega-tively correlated with testicular weight. at 10 mo of age.Mean tubular diameters in the mediastinuni region at90d were i06±7,98+7,89+6. and 78+6tmfor

100

.- 75CIO

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.-

0

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4; p. •. • •. ••'.•I '' Ø .I :: .

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Figure 3. Within testis divergence in anti-J\lullerian hormone (AMH) expression and diameter of seminiferous tubules of a 91-d-old crossbred(BX) hoar. These 2 fields were selected from the sanje section of tissue based on maximal divergence in degree of seminiferous tubule development.Expression of AMH was markedly greater in tubules with smaller diameters (A) than in tubules with larger diameters (B).

50

20

0<450 450 - 549 550 - 649 > 649

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Ford and Wise

ACID 0

Tubules near the tunica%o 040

G CP 0 000 o Negative for CDKNlB

30 0

d °o8 0 a Positive forCDKNlll

0001 0. 0 boo &A

000

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10 0 0

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OL

040 60 80 100 120 140

BTubules in the sub-tunica

• 40

0 0 Negative for CDKNIB0

10 £ Positive for CDKN1B

- 20

° °e10 0 0

A...- 0

40 60 80 100 120 140

Diameter of seminiferous tubule, .tm

Figure 4. The temporal relationship of anti-Mullerian hormone(AMH) with diameter in serial sections of individual seminiferous tu-bules located near the tunica (A) or in the sub-tunica (B) region of 6boars (?20 tubules/boar/region). Seminiferous tubules were classifiedas negative or positive for the presence of CDKNI B within the nu-clei of Sertoli cells. Each data point represents a specific seniiniferoiistubule that was evaluated for 3 characteristics: relative intensit y ofAMI-1 immnuno-st.aiuing, distinct presence of CDKN1B within Sertolicell nuclei, and diameter.

boars in the <450, 450 to 549. 550 to 694, and >649g testicular weight classes, respectively (data are notpresented for the sub-tunica region).

DISCUSSION

The present study established that onset of pubertaldevelopment in Meishan and BX boars was associatedwith loss of AMH iii time cytoplasm of Sertoli cells fol-lowed by expression of CDKN1B within Sertoli cell nu-clei. Both events occurred as diameter of seminiferoustubule began to increase. Expression of A'-\4H, a prod-net of less differentiated Sertoli cells, becomes estab-lished in boars by 28 d of gestation, continues throughgestation, and declines after parturition followed by anincrease at 2 to 3 wk of age (Tran et al.. 1977, 1981;McCoard et al., 2(02). Thereafter, expression, as deter-nuried by bioassay, decreases to nondetectable activityafter 60 d of age (Trait et al., 1981). In Meishan boats,AMT-I expression was less. as was its duration of ex-

p1Vi((m1. HII nt I ('F Hi )elviIt HI! (((liii )iln(i will! (!l

Testicular wt class, g

Figure 5. Classification of hours based oil weight at it)moo of age: boars were unilaterallcastrated at 7 (n = 20) or 9(1 (n =22) d of age. Distribution of boars aniong these classes differed withage at unilateral castration (P < 0.001).

expression of CDKN1B, a qualitative marker of Sertolicell maturation, reflects time earlier onset, of pubertal de-velopiiient in Meishan relative to the BX hours verify-ing earlier predictions (Harayamna et al., 1991: Lunstraet al., 1997; Kanematsu et al., 2006). An explanationfor the decreased expression of AMH at 7 and 28 d ofage in Meishan boars is not apparent. This differs fromsimilar expression of AMH observed in Meishan andcrossbred boars with onset of testicular differentiationand during early embryonic development (McCoard etal.. 2002). Germ cells are negative for AMH, therebyreducing the area within the seminiferous tubules thatexceeds the threshold of detection. but Meishan andcrossbred boats have comparable density of germ cellsin the first 3 wit of life (McCoard et. al., 2003). Longerexpression of A1'vi 1-1 in BX boars reflects the less maturestatus of their Sertoli cells and prolonged potential toproliferate in a portion of these cells. Suppression ofendogenous estrogen synthesis in prepubertal boats ex-ploits this proliferative potential, producing all increasein postpubertal testicular weight, uiore Sertoli cells,and greater sperni production (Berger et al.. 2008).

Expression of CDKN1B within Sertoli cell nuclei ofMeishan and BX boars was nondetectable. with thetechniques employed, at the youngest ages. Onset ofexpression of CDKN1B and its subsequent increase cor-related positively with expansion of seminiferous tubu-lar diameter in both genetic lines, signifying terminaldifferentiation as observed in rodents (Beumer et, al.,1999; Holsberger et al.. 2003). Expression of CDKN1Bin Sertoli cell nuclei occurred well in advance of senli-niferous tubules achieving their maximal diameter.

Increased androgen secretion by the early pubertaltestes marks onset of sexual maturity. Intratesticularandrogen concentrations have been nuplicated as a.negative regulator of At [IT secretion (Rev et ad.. 2003).I Iii piiia'ri:ii i'eint iu11I!i)) ( tl;il tin ('nih (Hat ii:ii:ii

lqq

(1i( 5(1 ((II (1 ii (hi 1(1(111 h(I lull

2541

period in boys and boars when elevated gonadotropniSecretions and increased testicular testosterone svii-thesis occur concurrent with elevated A1\IH secretion(Franca et al.. 2000: (Themes, 2001: Ford et al.. 2001). Alack of androgen receptors oilSertoli cells of bos dur-ing neonatal development (Clienies et al., 2008) offers ameans whereby Sertohi cells avoid the negative influenceof androgen on AMH expression. Then, at the onset ofpuberty. acquisition of 'androgen receptors within Ser-toll cells provides a link for suppressing AI\[H produc-tion. 1-lowever. this proposed cause and effect relation-ship is challenged in studies with mice that possess aSertoli cell-specific deletion of androgen receptor (Tanet al.. 2005). Their pattern of change iii AI\IH fromhigh expression at birth to low expression at pubertydoes not differ from control males. Likewise. froni birththrough puberty, these mice have an expression patternof CDl Ni B within Sertoli cells comparable with con-trol mice. In neonatal hoars, published reports disagreeregarding the presence of androgen receptors in Sertohicells (Kotula et al.. 2000: Ramesh et al.. 2007), leavingunresolved the role of androgens ill regulation of AMHproduction (luring early postnatal development.

The BX boars that were unilaterall y castrated at90 d of age had nearly a 3-fold difference ill testicu-lar weight, thereby providing the desired variation inonset of expansion of seuiimnferous tubules (initiationof pubertal development) for evaluation of sequentialchanges in AMII. CDKNI B. and seminiferous tubulediameter. Mean diameter of senuniferous tubules andpercentage of tubules containing Sertoli cell nuclei t hatdefinitively expressed CDKN I decreased with increas-ing distance froiii the niecliastinuni toward the tunica.indicative of a developmental gradient within the earlypubertal testis of boars. This relationship was unex-pected as it has not been reported in other species.Heterogeneity of AMI-T expression among tubules of put-bertal mice has been observed (Al-Attar et al., 1997:Mendis-Handagaina and Arivaratne. 2008), but a de-fined pattern was not reported. The observed patternof differentiation allows Sertoli cells iii the outer regionof the testes to proliferate for a longer period of time.Terminal differentiation of Sertoli cells occurs ill unisonwith formation of the blood-testis barrier and increasedsecretion of tubular fluids. With both ends of a senn-niferous tubule connect cr1 to the niediastinum. Sertolicell differentiation progressing outwardl y froni the me-diast inuni seems obligator y to allow fluids to flow to-ward the mnediastinum. Moreover, these observationssupport paracrinc regulation of Sertoli cells (lifferemitia-tion within a given tubule. In pubertal stallions, moreadvanced spermatogenesis occurs within the interior oftestes than within the outer region (Clenimuons et al.1995). It remains to be established if this out \vardlvprogressing pattern of seminiferous tubule differentia-tion is unique to pubertal boars and stallions.

Adjacent sections of the same seminiferous tubules(lefilied the sequence of change, with Ai\ IH production

decreasing before mmcii expansion of senmiuiferous tu-bules, followed by acquisition of CDKNIB in Sertohicell nuclei earlier than formation of a distinct lumen.Expression of CDKN1B in Sertohi cell nuclei and en-largement of seminiferous tubules are tightly linked innace (Holsberger et al, 2005). The CDKN1B classifica-tion of tubules was civahitative ; either no Sertohi cellsdefinitively expressed CDKN1B or some Sertohi cellsexpressed CDKNIB. Yet there was a rather clear de-marcation in the transition from less mature (increasedexpression of AI\IH and no detectable CDKN1B) tomore differentiated Sertoli cells (reduced AMII expres-sion and CDKN1B positive nuclei).

The consequence of the extent of Sertohi cell differ-entiation was apparent in the boars that were umnilat-erallv castrated at 90 cI of age. The subpopulation ofboars that developed the largest testicular weight at 10uio all had a low frequenc y of expression of CDKNIBin Sertoli cell nuclei at 90 ci and a mean seminiferoustubular diameter of less than 90 ji,lit iii the inecliasti-illilil region. These traits sigiufv ])oars predestined toundergo pubertal development at an older age amid, asa consequence, were able to elicit a greater coiimperisa-ory respomise to unilateral castration than boars with a

greater frequency of expression of CDKNIB in Sertohicell nuclei and semmmferous tubules of a larger diamim-eter. In contrast, the 23 of boars that were unilater-ally castrated at 90 ci of age but, developed time smmmahlest,testicular weight at 10 nmo all had tubule diametersgreater than 100 irm and increased frequency of CD-KN1B expression in their Sertohi cell nuclei supportingthe loss of proliferative potential of Serto!i cells anddevelopment of the blood-testis barrier. This latter sub-group attained pubert y at a younger age relative to theremainder of the boars. In marked contrast, none of theboars unilaterally castrated at 7 4 of age developed tes-ticular weight less than 450 g at 10 nio, indicating thatall had a full compensatory response corresponding totheir genetic potential.

The current studies document that AI\IH and CD-RN lB are markers of Sertoli cell maturity in boars andextend previous summaries of morphological develop-ment (Colenbra.iider et al. 1982: Franca et al.. 2005).Moreover. changes that signify terminal differentiationof Sertohi cells progressed fronm the nnediastinum towardthe tunica. Current knowledge regarding control of tes-ticular size of boars is incomplete. Consider the Meis-hiami amid Piau breeds: both have snmaller testes thanboars used in swine production, but these breeds dif-fer greatly in their rate of pubertal development, withMeishans being early, whereas Piau boars reach puber-ty at, a niucli older age (Franca et al., 2000). Contrastthese observations wit hi the studies in which direct se-lection for larger testes reduced pubertal age (Harder etal., 1995: Huang and .Johnson. 1996): thus. Sertohi cellproliferation of selected boars occurred more rapidlybefore the onset of terminal differential ion. producinglarger testes with greater sperm production than in

2542

Fort! and 1v1v15e

boars of the control line. Consequently, a clear under-standing of factors that interact to control daily spermproduction of boars does not exist; however, boars havea triore prolonged period of pubertal development thanrodents, thereby providing an excellent niodel to scru-tinize the interactions that determine the number ofSertoli cells that exist at sexual maturity.

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