Quntittive Determtion Follicle Size Distribution Gue Pig Ovry fter Hemicstrtion PMSG Tretment 1KATSUHIKO FUJIMORI, ROBERT M. NAKAMURA, SHARON A. TONETTA IsAO TSUTSUMI AND GERE S. dizerega Livgston Reproductive Biology Lbortory, Deprtment Obstetrics Gynecology, University Sourn Cliforni School Medice Los Angels, Cliforni 1Deprtment Obstetrics Gynecology Ymgt School Medice, Ymgt, Jpn Abst r ct In order to vestigte ction pot trphysiologicl or suprphysiologicl elevtion FSH durg preovultory period on folliculr development, dult gue pigs underwent unilterl ovriectomy on dys 10, 12 14 estrous cycle (N=6 ech group). Therefter, gue pigs jected twice dily with eir vehicle or pregnnt mre's serum gondotrop (PMS). After 2 dys, remg ovries removed. The resected ovries fixed, embedded prff, serilly sectioned (7ƒÊm) sted with Azn. All >70ƒÊm clssified by size tretic stge. The folliculr size distribution ws not ffected by hemicstrtion t dy 10, lthough rtio tretic (>400ƒÊm) decresed from 51% to 32% (P<0.01). Hemicstrtion t dy 12 cresed lrgest nontretic popultion (70-99ƒÊm group) from 17% to 26%, rtio tretic (>400ƒÊm) decresed from 35% to 23%. The pek size distribution ws shifted from 70-99ƒÊm to 200-299ƒÊm by PMS, 600-899ƒÊm size conted n cresed percentge tresi, which resulted bimodl distribution vible >400ƒÊm. These dt suggest tht 2 dy hemicstrtion promotes n flux primordil to growg suppresses tretic process by different mechnism dependg on dte hemicstrtion estrous cycle. Conversely, hemicstrtion+pms ccelerted vible follicle growth to crese percentge tresi. Received Jnury 6, 1988 Reprt Requests to: GERE S. dizerega, MD, Women's Hospitl, Room L 1013, 1240 North Mission Rod, Los Angels, CA 90033 This work ws supported prt by NICHD- HD 18601 The gue pig hve 5-6 dy preovultory period between corpus luteum demise ovultion durg 16 dy estrous cycle. As y ovulte 5-6 ov from biltrl ovries every cycle, 2 to 3 per ovry re selected from growg, remder re
FUJIMORI et l. dested to be tretic durg dys 12-16. Through preovultory period, elevted FSH ffects not only follicle development mture which escped tretic process ongog cycle but lso recruitment smll from follicle popultion sttic stte for next cycle. In this pper, to nlyze reltionship between extent elevtion FSH its effect on follicle development, hemicstrtion or hemicstrtion+pms ws designed s experimentl model trphysiologicl or suprphysiologicl elevtion FSH. First ll, we quntitted durg preovultory tervl estrous cycle gue pig to determe follicle popultions norml size distribution. Furr, we evluted ltertions this pttern distribution cludg tretic process fter hemicstrtion with or without PMS to elucidte which size rnge ws most FSH-sensitive by trphysiologicl or suprphysiologicl elevtion FSH. Mterils Methods Adult femle virg gue pigs (Dunk- Hrtly str), weighg 600-850g, housed under controlled temperture (24 Ž) with photoperiod 12hr (0600-1800hr) per dy fed Wyne R Gue Pig Diet (cludg vitm C) wter d libitum. Every morng (0900-1000hr), specific phses estrous cycle determed by spectg vgl membrne by vgl cytology (Young, 1938; Rwson et l., 1979 ; Grris Foremn, 1984). The first dy vgl openg (eir prtil or full) with presence nucleted /or cornified cells vgl lvge ws designted s dy 0 estrous cycle. All estrous cycles used for study immeditely preceded by t lest two consecutive regulr estrous cycles. Hemicstrtion ws performed lterntively by dorsl route under light er nessi t 1400-1500hr on dys 10, 12, 14 cycle. The excised ovries used s controls to estblish norml sequence folliculr development. Thirty nimls romly divided to two equl groups jected subcutneously with 0.2ml physiologicl sle or 5 IU PMS (pregnnt mre's serum, Sigm G 4877, Lot 114-0590-1, St. Louis, MO) 0.2 ml physiologicl sle. Ech niml received totl 4 jections : t 1800hr on dy hemicstrtion ws performed, t 1000hr 1800hr on next dy, t 1000hr on second dy fter hemicstrtion. After lst jection (1000hr), ll nimls scrificed by decpittion t 1600-1700hr. Immeditely fter decpittion, remg ovry ws dissected prepred for section while trunk blood ws collected, llowed to clot ser frozen for lter determtion hormone levels. Under sme protocol, hemicstrtion ws dditionlly performed on dys 11 13 to obt dy-by-dy hormone levels fter hemicstrtion t ech dy between dys 10-14. Intct gue pigs scrificed by decpittion sets 3 nimls per dy on dys 10-16 estrous cycle to collect norml serum ovries for morphometric nlysis. Quntittive Histology The ovries fixed Bou's solution dehydrted, embedded prff. Sections (7ƒÊm thick) serilly prepred sted with Azn. Every fifth section ws exmed by light microscopy, folliculr size degree tresi recorded for ll >70ƒÊm dimeter. The folliculr sizes, cludg c tern, determed by vergg two lrgest dimeters t right ngles dimeter through cross section contg nucleus oocyte usg clibrted occulr micrometer. A distction ws mde between without signs tresi tretic usg followg modifiction Willim's (1956) criteri (Rwson et l., 1979; Greenwld, 1973): Stge I: one lyer pyknotic nuclei nermost grnulos cell lyer contg t lest 5 cells. Or fdgs similr to those nontretic follicle. (Erly) Stge II: Up to 50% pyknotic nuclei grnulos distegrtion,
Vol.36, No.2 FOLLICULOGENESIS grnulos borderg ntrum.(mid) Stge III Almost complete distegrtion grnulos lyer (Lte) Folliculr tresi beyond stge III ws defed s which ntrl cvity ws completely replced by fibrocytic cells. These excluded from count s folliculr type could not be ccurtely determed. dys 10 12 cycle. Two nimls Gue pigs which hd cystic ovries lso excluded from study. The quntittive dt obted on cycle dys 10, 12, 14 estrous cycle from ovries first removed t time hemicstrtion from remg ovries to determe effects hemicstrtion (hemi) hemicstrtion plus PMS (hemi+pms). Hormone Assy Blood smples llowed to clot for 1 hr t 37 Ž. The serum ws collected fter centrifugtion t 2000rpm ~15m, stored t -60 Ž until estblished estrdiol, rostenedione, testosterone ssys could be performed (Mishell et l., 1971; Thorneycrt et l., 1973). Levels for ll smples ech hormone mesured sme ssy. Sttisticl Anlysis Dt evluted by rnkit or probit nlysis to evlute ir distribution expressed s men }Differences compred by Duncn's multiple rnge test P <0.05 ws considered to be significnt. Results The men cycle length gue pigs from this colony ws 16.0 }0.1 dy (n=80) vgl openg period ws 3.3 }0.1 dys (n=126). Spontneous ovrin cyclicity ws dicted by presence corpor lute from previous cycle ll nimls. There no differeces between groups s corpor lute (2.3 }0.2 per ovry), though 2 nimls hd unilterl ovultion lst cycle. The corpor lute mnifest morphologicl signs degenertions between
FUJIMORI et l. ovulted remg ovry fter hemicstrtion on dy 14. Quntittive Histology The totl nontretic ech size rnge (every 100ƒÊm) shown Tble 1. The percentge nontretic ech size rnge totl nontretic popultion is depicted Fig. 1. The distribution vrious dimeters control ovries from dys 10 through 14 demonstrted two popultions which converge t 400ƒÊm. The popultion <400ƒÊm occupied 87% totl, while modl dimeter ws 250ƒÊm for dys 10 through 14 (Fig. l). At dy 14, reltive percentge 200-299ƒÊm dimeter slightly Fig. 1 Fig. 1. The normlized men percentge vible gue pig ovry more thn 70ƒÊm dimeter. : untreted ovry (estrous cycle dys 10, 12 14) Fig. 1 b b: ovries from estrous cycle dy 14 (untreted control, fter hemicstrtion fter hemi+pms)
Vol. 36, No. 179 FOLLICULOGENESIS 2 num 70-99μm cresed group l D A Y12 On dy 12 fter hemi or hemi+pms, distribution folliculr sizes ws similr to control. On dy 14 (Fig. lb), percentge smll 70-99 pm cresed to 17% while modl dimeter remed 250 μml DA Y14 hemi+ PM S :u ntr e ted ovr E str o usc y c le d y 14 o vr y;u n tr et ed, f ter he mi ndf te r cles(> 40 0μmi nd im eter) p e r3o f terh em i +PMS ll i i) e cum ultiv enu mb v ries. y(est rouscycl edys e r g u i nepig fo Fig. 2.Th b : Estrous c ycl e dy 1 2o vr y ; u ntrete d, fterh emic st rt i on( h em decresed while ber In ddition, re 10,121 4) ws n crese percentge 7099 pm dimeter from 17% control group to 27% (P<0.05) hemicstrtion group where pek size ws 70-99 pm. In contrst, folliculr size distribution hemi+pms group conted modl dimeter 150 pm. Fig. 2 shows cumultive tretic (stge I+ 1I+1I1) nontretic > 400 pm for control groups on dys 10, 12 14. The ctul s recent tretic (stgel+ II) control group constntly 17 or 18 per 3 ovries on dys 10, 12 14. The nontretic ws decresed on dy 12 compred to dys 10 14. Fig. 2b shows observed t dys 12 14 for norml, hemi, hemi +PMS group. On dy 12 hemi,
180 FUJIMORI vible cresed with no chnge tretic. After hemi+pms, stge III tretic significntly cresed (P<0.05) from 11 hemi group Fig. : to 30 per 3 ovries 3. The represnts untreted 14) size distribution cumultive ovry (estrous Dy 10(n=7) Dy 12(n=7) Dy 14(n=B) with no chnge dys 10, 12 Jpon. vible. On dy 14 hemi, recent tretic (stge I+II) decresed from 17 to 6 per 3 ovries while nontretic lte tretic (stge III) ws similr. On dy 14 fter ddition PMS to gue pig per cycle Endocrol. April 1989 et l. b: more thn 400pm 3 ovries ech dimeter. size ctegory Ech Estrous cycle dy 12 ovry (untreted, hemi fter hemi+pms) DAY 12 br. fter
Vol. 36, No. 181 FOLLICULOGENESIS 2 hemi, remg ovry conted significnt crese (P<0.05) recent tretic with slight decrese nontretic. The percentge tretic/totl, clculted from cumultive s, showed significnt decrese hemi group when compred with vehicle-treted dy controls 12, 14, P<0.05; Figs s 3, b (untreted, fter gue nontretic 10 size distribution. (Fig dy On 60%) observed The 400-490ƒÊpm >900ƒÊm. which itition tretic folliculr sme ws evident ws especilly (Fig. 3c). distribution dys vible with 12 tretic rnges on dys 12 hemi group, 800ƒÊpm This fter ws cycle. I+II) group. 14 mimum (stge dy 3b). (P<0.05) 700 generlly (Fig. hemi+pms 14 800-899ƒÊm controls on bimodl (>400ƒÊm) 600-799ƒÊm In dimeter ws decrese III s. ovries evident for 400ƒÊm control tretic lrgest group mrked size tretic Stge distribution I 600-899ƒÊm 500-700ƒÊm size s found hemicstrted rnge contrst, contg However, be ctegory, common In rnge The tresi. with size group to I frequently Stge limited (50- size suggests 400 Stge most 600-799ƒÊm detected rnge from rnge. rrely mximum obsig- proportion ws ws 600ƒÊm. 500-799ƒÊm puttive observed dimeter. tretic on when II identicl nontretic 400 tretic/totl between pm 14, (>1000ƒÊm) time, re crese 900 In lmost dy first histogrms (>400ƒÊm) 3), 12. Atretic s t t pig (P<0.05) to c represent nificnt 32.2% 22.8% function norml DAY 14 preovultory served for Estrous cycle dy 14 ovry hemi fter hemi+pms) vs vs P<0.05). dys c: (44.5% 35.3% found to groups on The 700-799ƒÊm lmost 14, respectively. sme Hormone Levels The men concentrtions estrdiol, rostenedione testosterone determed on dys 10-16 estrous cycle re
FUJIMORI et l. Endocrol. April 1989 Jpon. depicted Fig. 4. In vehicle-treted nimls, estrdiol vlues grdully cresed from 6.6 }2.2pg/ml t dy 10 peked t 25.7 } 5.0pg/ml on dy 15. Estrdiol levels serum fter hemi mted pttern which ws prllel to estrdiol Fig. 4. Men serum concentrtions estrdiol (pg/ml), rostenedione (pg/ml) testosterone (pg/ml) throughout estrous cycle dys 10-16 gue pig under untreted, hemi, heim+pms (N=3-5 nimls per plot.) levels controls lbeit shifted 1 dy from control nimls. After hemi+pms, estrdiol levels significntly cresed from bsl vlues through dys 12-14 (P < 0.01), peked (37.0 }9.1pg/ml) t dy 13 grdully decresed on dys 14-16. Both rostenedione testosterone concentrtions significntly decresed fter hemi (P<0.05) significntly cresed fter hemi+pms comprison with control nimls (P<0.05). Discussion The present study reveled tht shortterm (2 dy) hemicstrtion durg folliculr growth (dy 10-14) cclerted flux <70ƒÊm dimeter to smll prentrl reduced rte tresi >400ƒÊm. Subsequently, concomitnt PMS shifted folliculr size distribution towrd lrger compred to hemicstrtion group. PMS ppered to ccelerte growth 70-99 from <70ƒÊm. Therefore, 70-99ƒÊm dimeter re sensitive to creses FSH. Sce only tretic stge I+ II is bruptly decresed hemicstrtion group (on dy 14) compred with or groups, hemicstrtion seems to hibit itition tresi or decelerte tretic process grnulos cells t stge 1+11. Hemicstrtion (on dy 12) enhnced s lrge nontretic to reduce reltive percentge tretic/ totl compred to tct controls. Thus, mechnism for crese percentge nontretic hemi group pper to depend upon extent folliculr mturtion t time hemicstrtion is done. In ddition, more modest elevtion gondotrop levels fter 2 dy hemicstrtion might fcilitte folliculr mturtion only physio-
Vol. 26, No. 2 FOLLICULOGENESIS logicl mnner. This is lso supported by steroid vlues present study. As shown Fig. 4, one dy ws required fter hemicstrtion for elevted gondotrops to restore production estrdiol to norml level. Therefore, two dy hemicstrtion ws considered to be pproprite for trphysiologicl elevtion gondotrops our experimentl design. Hermreck Greenwld (1964) reported tht dy 12 seems to be criticl period for compenstory response (twice Grfin >800pm dimeter twice mount tresi this size rnge) fter hemicstrtion gue pig. In ir study, period hemicstrted condition ws vrious longer thn 2 dys becuse ll remg ovries exmed on dy 1 next cycle hemicstrtion. Our dt suggest tht two dy hemicstrtion ffects tretic process even fter dy 12. More detiled formtion would be required to underst reltionship between effect 2dy hemicstrtion longer hemicstrtion. After hemi + PMS tretment, serum estrdiol, rostenedione teststerone levels rose, presumbly suprphysiologicl response to dditionl gondotropic stimultion (Fig. 4). Hemi + PMS did not result higher percentge nontretic, fct, prdoxiclly cresed percentge tretic on dy 14 compred to untreted controls (Fig. 2b). In present study, 5 IU PMS hemicstrted gue pig seemed to fcilitte ongog tresi remg ovry. Likewise, duction tresilike processes by exogenous PMS ws reported by Peluso (1979), Blchrn et l.(1983) Ryn (1981). However, Fig. 3, bimodl distribution nontretic becme pprent with n flection pot 800-899pm. Furr, hemi + PMS tretment ws ssocited with decrese vible with no ltertion s tretic with rnge 800 to 899 pm on dys 12 14. Therefore, this modultion might be result n cresed vible pssg through 800-899pm size rnge. Although hemi ws ssocited with generlized reduction tretic ll size clsses, effects co-dmistered PMS confed to 800-899pm group, dictive gondotrop-sensitive selection process extnt prior to tht stge folliculr mturtion. The tretic process, refore, is lso gondotrop-sensitive event which cn vry with degree elevtion gondotrop levels. We found which develop beyond primordil stge gue pig ovry to be 100-280 regrdless dy estrous cycle, reby confirmg previous reports (Myers et l., 1936; Dempsey et l., 1937; Schmidt, 1942; Lbhsetwr Dimond, 1970; Peddie, 1980, b; Grris Foremn, 1984). Although ctul s vried considerbly between dividul ovries, chnges percentge distribution folliculr size consistent ovries throughout cycle dys 10-14. As whole, re two distguishble popultions, distribution ptterns which converged t 400pm dimeter. The popultion 70-399pm dimeter represented 87% totl popultion, wheres rest > 400pm dimeter. The convergg pot 400pm ws mted even fter hemicstrtion with/without PMS present study. Previous vestigtors (Schmidt, 1942; Hermreck Greenwld, 1964) reported tht most > 400pm become tretic by dys 3 fter ovultion, levg only nontretic <400pm 4-5 dys fter ovultion. Thus, we recognize tht 400 pm cn be regrded s n isosbestic selection pot folliculr development.
FUJIMORI et l. Endocrol. April 1989 Jpon. The lrgest group hd dimeter 200-299 pm which comprised 35% totl. The cl lyer could be distguished morphologiclly conjunction with ppernce ntrl cvity, round 200ƒÊm dimeter. Anlysis folliculr size distribution reveled two groups < 400ƒÊm, one 70-99ƒÊm dimeter, or 150-3 50ƒÊm dimeter. A shift pek percentge distribution from 200-299 to 70-99ƒÊm dimeter group ws found to ccompny hemicstrtion, but not fter PMS rpy. Hemicstrtion, with or without PMS, clerly cresed 70-99 pm dimeter by dy 14 estrous cycle, suggestg tht this size / or type follicle conted gondotropresponsive group from which recruitment occurs. Sce Fig. 3 presents nerly sme distribution vrious size ctegories between dys 10 12, vible do not pper to rise from popultion < 400ƒÊm dimeter durg this period. Rr, ctul s vible showed rpid crese from dy 12 to dy 14 despite no chnge tretic (Fig. 3). Thus, flux vible from popultion < 400ƒÊm ppered to be ccerelted between dys 12 14 estrous cycle, which ws remiscent effect hemicstrtion (on dy 12) s mentioned bove. The size where tretic chnges becme common ws 600-799ƒÊm, suggestg tht size selection, from pot view tresi, occurs with dimeters < 600ƒÊm, which both confirms extends fdgs obted by Peddie (1980) prepubetl gue pig ovry. Stge III tretic more frequently encountered 400-699ƒÊm group contrst to 600-799ƒÊm size typicl with morphologicl evidence recent tresi (stge I+ II). In this respect, tretic process my first become morphologiclly evident 600-799ƒÊm dimeter, n become rrested by loss grnulos lyer be followed by collpse ntrum (400-699ƒÊm). Tkg ll results here toger to considertion, four selection pots cn be identified folliculr development durg estrous cycle gue pig: 1) primordil <70ƒÊm, 2) 200ƒÊm, dimeter which is cocident with ntrl formtion ppernce c, 3) isosbestic convergg pot 400ƒÊm, 4) 600-799ƒÊm dimeter which mnifests n enhnced sensitivity to tresi.. Acknowledgments The uthors wish to thnk Dr. Anne Hirshfield for her ssistnce terprettion dt Ms. Ky Moon for her timely preprtion mnuscript. References Blchrn, P. K., S. B. Moodbidri T. D. Nekr (1983). Autordiogrphic nlysis FSH bdg durg folliculr tresi. Experimenti 39, 792-793. Butcher, R. L.(1984). Pttern folliculr growth durg four dy estrous cycle rt. Biol. Reprod. 31, 280-286. Dempsey, E. W.(1937). Folliculr growth rte ovultion fter vrious experimentl procedures gue pig. Am. J. Physiol. 120, 126-132. Grris, D. R. D. Foremn (1984). Folliculr growth tresi durg lst hlf lutel phse gue pig estrous cycle: Reltion to serum progesterone estrdiol levels utero-ovrin blood flow. Endocrology. 115, 73-77. Greenwld, G. S.(1973). Quntittive spects folliculr development untreted PMS-treted cyclic hmster. Ant. Rec. 178, 139-144.
Vol. 36, No. 2 FOLLICULOGENESIS Hermreck, A. S. G. S. Greenwld (1964). The effects unilterl ovriectomy on folliculr mturtion gue pig. Ant. Rec. 148, 171-176. Hirshfield, A. N. A. R. Midgley, Jr.(1978). Morphometric nlysis folliculr development rt. Biol. Reprod. 19, 597-605. Lbhsetwr, A. P. M. Dimond (1970). Ovrin chnges gue pig durg vrious reproductive stges steroid tretments. Biol. Reprod. 2, 53-57. Mishell, D. R., R. M. Nkmur, P. G. Crosignni, S. G. Stone, K. Khrm, Y. Ngt I. H. Thorneycrt, (1971). Serum gondotrop steroid ptterns durg menstrul cycle. Am. J. Obstet. Gynecol. 111, 60-65. Myers, H. I., W. C. Young E. W. Dempsy (1936). Grfin follicle development throughout reproductive cycle gue pig, with especil reference to chnges durg oesterous (sexul receptivity). Ant. Rec. 65, 381-401. Peddie, M. J.(1980). Folliculr development immture gue pig. J. Endocrol. 84, 323-331. Peddie, M. J.(1980b). Folliculr development dult gue pig response to humn chorionic gondotrop. J. Endocrol. 85, 9-16. Peluso, J. J.(1979). In vitro mturtion oocytes collected from tretic. In: Ovrin Folliculr Developement Function. Midgley AR, Sdler WA (eds.). New York, Rven Press, pp 85-88. Rwson J. M. R., C. I. Gley, L. C. Weberg B. J. Hodgson (1979). Effects gondotrops on follicullr development, ovultion tresi mture gue pig. Hormone Res. 10, 25-36. Ryn, R. J.(1981). Folliculr tresi: Some specultions on biochemicl mrkers mechnism. In: Dynmics Ovrin Function. Schwrts, NB, Hunzicker M, Dunn (eds.). New York; Rven Press, pp 1-12. Schmidt, I. G.(1942). Mitotic prolifertion ovry norml mture gue pig treted with colchice. Am. J. Ant. 71, 245-270. Thorneycrt, I. H., W. O. Ribeiro, S. C, Stone S. A. Tilson (1973). A rdioimmunossy rostenedione. Steroids. 21, 111-118. Willims, P. C.(1956). The history fte redundnt. Cib Foundtion, Agg, 2, 59-68. Young, W. C.(1938). The vgl smer picture, sexul receptivity time ovultion gue pig. Ant. Rec. 67, 305-325.