Developmental stages of fetal-type Leydig cells in prepubertal rats
T. KUOPIO1*, J. TAPANAINEN2, L. J. PELLINIEMI3 and I. HUHTANIEMI4
Departments of Anatomy^ and Physiology*, and Laboratory of Electron Microscopy*, University of Turku, Turku, and Department ofObstetrics and Cynecology2, University of Oulu, Oulu, Finland
* Address for reprints: Department of Anatomy, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland
Summary
Fetal Leydig cells were studied in rats during and afterthe perinatal-neonatal period by comparing changes inmorphology, number and volume with changes in tes-ticular steroids and serum luteinizing hormone (LH)concentration. Stereologic examination indicated re-gression of fetal Leydig cells in testis by showing thattheir total volume as well as the average cell volumedecreased between prenatal day 20 and postnatal day 3.The total number and total volume of cells bothincreased between postnatal days 3 and 11 but theaverage cell volume did not change during the same timeperiod. Determination of serum LH showed a closecorrelation between an increase in LH concentration andincreases in total number and volume of cells. Thecombined number of fetal- and adult-type Leydig cellson day 20 was more than 20 times the number of fetalcells at 3 days of age. Electron microscopic analysisshowed that fetal Leydig cells after birth formed con-spicuous clusters, which were surrounded by a layer ofenvelope cells and extracellular material. Occasionaldividing fetal Leydig cells and possible precursors offetal or adult Leydig cells were observed. Mitoses ofspindle-shaped pericordal cells were frequent during theneonatal period. During and after the second postnatal
week fetal Leydig cells again showed signs of regression,indicated by disintegration of the cell clusters, a decreasein cell size, accumulation of collagen between the cellsand a decrease in steroid content per cell. The cytoplasmshowed no degenerative changes, but the shape of thenuclei changed from spherical into irregular.
The present results suggest that the perinatal re-gression of fetal Leydig cells is followed by an LH-induced phase of growth. This growth period precedes asecond phase of regression that coincides with earlydevelopment of adult-type cells reported earlier. Con-trary to the present concept of a biphasic pattern ofLeydig cell development in rat, we suggest threeconsecutive stages: fetal (fetal cells in fetal testis), earlyjuvenile (fetal cells during neonatal-early juvenile life)and juvenile-adult (adult cells before and after pu-berty). The regression of the fetal cells suggests that theyhave only a minor role in testicular hormone productionafter the appearance of adult-type Leydig cells prior topuberty.
Morphologically and functionally distinct populationsof fetal and adult Leydig cells are present in testis ofdeveloping rats (Christensen, 1975; Huhtaniemi et al.1984; de Kretser and Kerr, 1988). The cells of thepopulations are referred to as fetal and adult Leydigcells, because they differentiate and start their hormoneproduction during fetal life and before and after pu-berty, respectively. It has been suggested that fetalLeydig cells decrease in number and finally disappearfrom the interstitium as a result of cell death ordedifferentiation during postnatal life (Gondos, 1977).However, recent studies have indicated that the totalnumber of the cells in testis does not change markedlyimmediately after birth even though the number of thecells per unit volume of testis decreases (Mendis-
Handagama et al. 1987; Zirkin and Ewing, 1987; Kerrand Knell, 1988; Kuopio et al. 1989a). Moreover, Kerrand Knell (1988) recently reported their intriguingfinding that fetal Leydig cells persist as a distinctpopulation in adult testis after the growth of adult-typecells.
Earlier we showed that testicular steroid content perLeydig cell is highest during fetal life, suggesting thatfetal Leydig cells are steroidogenically more active thanadult-type cells (Tapanainen et al. 1984). Before birth,the steroid content per cell decreases by an unknownmechanism but still remains higher than that in adultsthrough the first and second postnatal weeks (Tapan-ainen et al. 1984).
The present study extends our earlier findings(Tapanainen et al. 1984; Kuopio et al. 1989a,b,c) ofmorphological and functional differences in fetal Ley-
214 T. Kuopio and others
dig cells before adult-type cells begin to dominate. Ourresults explain the discrepancy between the old conceptof early Leydig cell regression and recent studiesreporting a constant number of cells in pre- andpostnatal rats. Moreover, our results suggest a luteiniz-ing hormone-induced proliferation of the fetal cellsfollowed by their structural and functional regressionafter the early neonatal period.
Materials and methods
Animals and specimen preparationMale Wistar rats between fetal day 20 and postnatal day 23were used for this study. The animals were kept in controlledtemperature (22 °C) and photoperiod (14L:10D). Laboratoryanimal chow and water were available ad libitum. The ratswere killed by decapitation, blood was collected for serumluteinizing hormone (LH) measurements and testes weredissected out, weighed and prepared for morphologic andstereologic analysis. The testes were fixed by immersion in5 % glutaraldehyde in 0-16% moll"1 2-, 4-, 6-collidine-HClbuffer (pH7-4) and further processed as described earlier(Kuopio et al. 19896). Sections (l^rn) were stained withtoluidine blue for morphometry at the light microscopic level(day 20 of gestation and days 3, 11, 15 and 20 postnatal).Sections for electron microscopy (day 20 of gestation to day 23postnatal) were stained with uranyl acetate and lead citrate.Specimens were collected for analysis of testicular steroids atdays 3, 11 and 15 after birth as described earlier (Tapanainenetal. 1984).
Stereologic methodsStereologic measurements included only the fetal type Leydigcells except at 20 days postnatal, when adult-type cells wereincluded to analyse the total number of Leydig cells of bothtypes per testis. Cells were identified according to criteriadescribed earlier (Mendis-Handagama et al. 1987; Zirkin andEwing, 1987; Kerr and Knell, 1988; Kuopio etal. I989a,b,c).The stereologic methods of the present study have beendescribed earlier (Kuopio et al. 1989a). Briefly, the volumedensities of Leydig cells (VVLC> fraction of testis volumeoccupied by the cells) and Leydig cell nuclei (VV Nuc) wereestimated using the point-counting method (Weibel, 1979).The numerical densities of Leydig cells ( N V L C . number ofcells per unit volume of testis) were calculated from V V N ucand from the number of nuclear profiles of Leydig cells perunit area of section (NA N Uc) using the method of Weibel andGomes where N V L c = l / / 3 * ( N A N U C
3 / 2 / V V N U C 1 / 2 ) - Thecoefficient yS is a dimensionless shape coefficient, which, in thecase of nearly spherical particles, =1-38 (Weibel, 1979). Tocalculate the total volume and number of Leydig cells pertestis, VVLC and NVLC were multiplied by testis volume,respectively. Testis volume was obtained directly from weightmeasurements because the specific gravity of testis does notconsiderably differ from l-0gcm~3 (Mori and Christensen,1980). Testicular weights and numbers of animals used in thestereologic analysis are shown in Table 1. The average volumeof Leydig cells was derived by dividing V V L C by N V L C -Measurements were made from three randomly selectedblocks from each animal and from one randomly selectedl|imi section from each block. The whole section area wasanalyzed using a microscope with a 40 x objective lens and aneyepiece grid. The grid covered 0-02 mm2 at a time and had625 line intersections. The average number of analysed fieldsper animal was 100.
Table 1. The number of animals and the weights ofthe testes (mean ± S.E.M.) used in the stereologic
measurements
Age (days) Number of animals Testis weight (mg)
f20* 53 5
11 515 420 4
* days of fetal life (f).
1-5 ±0-12-910-320 ± 0-250 ±1-0
110 ±5
Determination of serum LH concentration and steroidcontent per Leydig cellSerum LH was measured with homologous radioimmuno-assay kits provided by The National Pituitary Agency andNIADDK (Bethesda, MD) as described earlier (Huhtaniemiet al. 1986). Results are expressed in terms of the RP-1standard. To estimate the total steroids per Leydig cell thesteroid content per testis was calculated from measurementsof individual steroid concentrations (testosterone, 5a'-dihy-drotestosterone, progesterone, 17-hydroxy progesterone,pregnenolone, androstenedione and 5<r-androstane-3a-, 17/3-diol) published previously (Tapanainen etal. 1984). Values ofthe steroid content per testis were then divided by the numberof cells per testis (Fig. 3) from randomly selected pairs inrespective age groups of animals. The obtained individualvalues were averaged and analyzed statistically.
StatisticsStatistical comparisons of the age groups were made usingNewman-Keuls multiple range test (N-K).
Results
Leydig cell stereologyThe total volume of fetal Leydig cells in testis (Fig. 1)and the average Leydig cell volume (Fig. 2) decreased(P<0-05) in the perinatal period. The total number ofcells did not significantly change during the same time(Fig. 3). Between days 3 and 11 after birth, the totalnumber (Fig. 3) and volume (Fig. 1) of cells increasedsignificantly (P<0-05). The combined number of the
f20 3 11 15Age (days)
Fig. 1. Total volume of fetal Leydig cells from fetal day 20(f) to postnatal day 15. Each bar is the mean ± S.E.M. of 4to 5 animals. Statistically significant differences wereobserved between groups indicated by different letters
-05, N-K) .
Prepubertal fetal Leydig cells 215
fetal- and adult-type Leydig cells on day 20 was morethan 20 times the number of the fetal cells at the age of 3days (Fig. 3). No significant change was seen in averagecell volume between ages of 3 and 15 days (Fig. 2).
Leydig cell morphologyAfter birth, fetal Leydig cells formed conspicuous,
smoothly delineated clusters in which the cells weretightly packed and intercellular spaces were narrow.The clusters were surrounded by a layer of envelopecells and extracellular material which separated themfrom the surrounding interstitium. No other cell typesOr blood vessels were found inside the clusters (Fig. 4).
Cell number (cells x 104/testis VZQ)(cells x 105/testisCZ])
GO 3 11Age (days)
Fig. 2. Average fetal Leydig cell volume from fetal day 20to postnatal day 15. Each bar is the mean ± S.E.M. of 4 to 5animals. Statistically significant differences were observedbetween the groups indicated by different letters (f<0-05,N-K).
3 11 15Age (days)
Fig. 3. Total number of fetal Leydig cells in testis fromfetal day 20 to postnatal day 20. Each bar is themean ± S.E.M. of 4 to 5 animals. Statistically significantdifferences were observed between the groups indicated bydifferent letters (P<0-05, N-K).
, •
VfWli.
f VL
L
Fig. 4. Electron micrograph of a cluster of fetal Leydig cells (L), age 8 days. Cells in the cluster are closely attached to eachother. The cluster is surrounded by an envelope cell (e). The space between the Leydig cells and the envelope cell containspositively staining collagen fibers (thin arrows). x4500.
216 T. Kuopio and others
• %
c *
Fig. 5. Electron micrograph of two pericordal cells in mitosis (thick arrows), age 5 days. The cell on the left is immediatelyadjacent to the testicular cord (c) while the one on the right is separated from the cord by one or two cell layers. X4500.
Mitoses of spindle-shaped pericordal cells were fre-quent during the early days of-life (Figs 5 and 6).Occasional mitoses of fetal Leydig cells (Fig. 7) andimmature cells without typical features of Leydig cellsor mesenchymal cells (Fig. 8) were also observed in theinterstitium.
During and after the second postnatal week, someclusters of fetal Leydig cells started to disintegrate. Thecells in the clusters were separated from each other byintercellular spaces which increased in size and becamefilled with negatively staining collagen fibers (Fig. 9).The expansion of the space between the cells probablyoccurred as a consequence of reduced cell size. Therewere no degenerative changes in the cytoplasm, but thecharacteristically spherical nuclei (Fig. 4) became ir-regular in shape (Fig. 10). Interstitial macrophageswere often associated with clusters that showed thistype of disintegration (Fig. 10).
Serum LH concentration and steroid content perLeydig cellA significant increase in serum LH concentration wasseen between the days 3 and 11 (Fig. 11). The change inthe LH concentration significantly (P< 0-001) corre-lated with an increase in total number (r = 0-9969) andvolume (r = 0-9899) of fetal Leydig cells during thesame period (days 3 and 15), suggesting a cause-and-effect relationship. Steroid content per Leydig cellsdecreased significantly between days 11 and 15(Fig. 12). The correlation between steroid content per
cell and serum LH concentration was negative(r = 0-9057) and statistically significant (P<0-02).
Discussion
Fetal Leydig cells are generally believed to decrease innumber and finally to disappear from the interstitiumduring a process of regression after birth (Gondos,1977). The present study and other recent reports,however, suggest that the total number of fetal Leydigcells in testis does not change significantly between theend of pregnancy and the early days of postnatal life(Mendis-Handagama et al. 1987; Zirkin and Ewing,1987; Kerr and Knell, 1988; Kuopio etal. 1989a). At thesame time, the present results confirm and extend theestimate of Roosen-Runge and Anderson (1959) whoreported that total Leydig cell volume decreases be-tween the late fetal period to the fourth postnatal day.If the total number of cells does not change significantlyduring the perinatal period, then the decrease in totalvolume of cells must be due to a reduction in size ofindividual cells. This conclusion has been supported byrecent observations of Zirkin and Ewing (1988) andconfirmed by us here. Earlier reports of drastic Leydigcell regression after birth (Roosen-Runge and Ander-son, 1959) may be explained by a marked decrease innumerical density, or the number of the cells per unitvolume of testis (Lording and de Kretser, 1972;Tapanainen et al. 1984; Zirkin and Ewing, 1987; Kerr
Prepubertal fetal Ley dig cells 217
Fig. 6. Light micrograph of several testicular cords with two pericordal cells in mitosis (thick arrows), age 5 days.c, testicular cord. x310.Fig. 7. Light micrograph of a fetal Leydig cell (arrowhead), and a pericordal cell (thick arrow) in mitosis, age 1 day.c, testicular cord. x610.Fig. 8. Electron micrograph of a group of interstitial cells which are considered immature fetal or adult Leydig cells, age 5days. X4500.
and Knell, 1988) as well as a reduction in individual cellvolume. In addition, the reorganization and clusteringof fetal Leydig cells soon after birth (Roosen-Rungeand Anderson, 1959; Kuopio et al. 1989c) may give animpression of regression.
The decrease in total volume of fetal Leydig cellsduring the perinatal period is followed by a postnatalphase of regrowth, also observed by Roosen-Runge andAnderson (1959) and Mendis-Handagama et al. (1987).According to the present study, this growth phase isrepresented by an increase in the number of Leydigcells, not from changes in cell size as is true during theperinatal phase of regression. Similarly, Mendis-Handagama et al. (1987) found an increase in thenumber of the cells between the days 5 and 10, whereasKerr and Knell (1988) reported no comparable changeduring the first and second postnatal weeks. Anotherdiscrepancy in earlier literature concerns the ratio ofLeydig cell number just after birth to that during andafter the third postnatal week. Our earlier report of alarge decrease in steroid content of Leydig cells fromperinatal period to adulthood was based on measure-ments showing a 20-fold increase in Leydig cell numberbetween days 2 and 3 versus 3 weeks of age (Tapanainenetal. 1984). Recently, Zirkin and Ewing (1987) reportedonly a fivefold increase in cell number during the
corresponding period, a result that would negate ourearlier conclusions reporting differences in steroid pro-duction of fetal versus adult cells (Tapanainen et al.1984). The findings in the present study, however, are inaccord with our earlier results (Tapanainen et al. 1984)and also supported by data of the Leydig cell numbersfrom the other laboratories (Mori and Christensen,1980; Mendis-Handagama et al. 1987; Kerr and Knell,1988).
The direct correlation between an increase in serumLH and the number of cells per testis suggests that thepostnatal growth phase of the fetal Leydig cells may beregulated by a physiological rise in serum LH during thefirst week of life (Lee et al. 1975; Ketelslegers et al.1978; Ramaley, 1979). This idea receives support fromour earlier study which showed a marked and rapidincrease in the number of fetal Leydig cells in newbornrats after exogenous hCG administration (Kuopio et al.1989a). The presence of mitoses in fetal Leydig cells ofuntreated rats and earlier in rats treated with hCG(Kuopio et al. 1989a) suggests that cell proliferation cancontribute to the increase in cell number. Fouquet andKann (1987) emphasise that convincing evidence formitoses in Leydig cells in young rats is not availablebecause the reports are based on observations onparaffin sections from which identification of cells is not
218 T. Kuopio and others
- / - i ^ \
10
Fig. 9. Electron micrograph of a cluster of fetal Leydig cells (L), age 15 days. The cells are separate from each other, andnegatively staining collagen fibers (thin arrows) appear in the intercellular spaces. X4400.Fig. 10. Electron micrograph of a regressing cluster of fetal Leydig cells (L), age 23 days. The cytoplasm of the cells isreduced and the intercellular spaces are large. A macrophage (m) is in a typical location close to the regressing cluster,c, seminiferous tubule. x3800.
3 11 15Age (days)
Fig. 11. Serum LH concentration in rats at ages 3, 11 and15 days of postnatal life. Each bar is the mean ± S.E.M. of 4to 5 animals. Statistically significant differences wereobserved between groups indicated by different letters(><0-05, N-K).
3 11 15Age (days)
Fig. 12. Mean content of total steroids per fetal Leydigcells at the ages of 3, 11 and 15 days of postnatal life. Eachbar is the mean ± S.E.M. of 3 to 5 values. Statisticallysignificant difference was observed between the groupsindicated by different letters (P<0-05, N-K).
reliable. The same criticism cannot be applied to thepresent observations because mitotic figures can bereliably identified in 1 ,um plastic sections. In addition tocell proliferation, differentiation from immature pre-cursors may increase the Leydig cell number as well. Atthis time, however, it is not possible to differentiatebetween precursors of fetal Leydig cells versus adultLeydig cells. Therefore, the contribution in numericalgrowth of the two cell types made by differentiation ofimmature interstitial cells and that made by mitotic cellswith an elongated shape in the pericordal positionremains to be clarified.
The postnatal increase in fetal Leydig cell number issoon followed by a phase of regression. This is indicatedby disintegration of cell clusters, accumulation of col-lagen between the cells and a decline in the steroidcontent per cell. Earlier we showed a decrease insteroids per cell during the last days of fetal life andagain after day 20 (Tapanainen et al. 1984). The latterdecline coincided with replacement of fetal Leydig cellsby an adult cell population and therefore probablyrepresented a difference in the steroidogenic capacity ofthe two cell populations (Tapanainen et al. 1984). Thepresent analysis, directed in more detail to the first and
Prepubertal fetal Leydig cells 219
second postnatal weeks, showed in addition, a declinein steroids per cell preceding the shift between the twopopulations. This decline coincides with a small de-crease in serum LH concentration during the secondpostnatal week observed previously (Lee et al. 1975;Ketelslegers et al. 1978; Ramaley, 1979), but not in thepresent material. Deprivation of LH would be consist-ent with functional changes in fetal cells. However, invitro testosterone production per testis in the presenceof maximally stimulating amounts of hCG decreasesbetween days 5 and 10 (Huhtaniemi et al. 1982) despitea simultaneous increase in total cell number. Thissuggests that changes in serum LH concentration be-tween the days 11 and 15 per se may not be responsiblefor the observed in vivo decrease in the steroids per cell.Moreover, LH receptor measurements (Huhtaniemi etal. 1982) do not indicate that changes in number ofreceptors can explain decreased steroid production percell during the second postnatal week. The physiologicrole of the small population of fetal Leydig cellspossibly persisting after puberty (Kerr and Knell, 1988)remains to be investigated. As far as hormone pro-duction is concerned, they appear to be an unimportantminority when compared with the adult-type cells,which may be more than 200- to 500-fold greater innumber (Mori and Christensen, 1980; Tapanainen et al.1984; Mendis-Handagama et al. 1987; Kerr and Knell,1988).
Our study showing disintegration of Leydig cellclusters and accumulation of collagen fibers in expand-ing intercellular spaces is consistent with an earlyhistologic study (Roosen-Runge and Anderson, 1959),in which the Leydig cell clusters were seen to bedispersed into individual cells separated by a densenetwork of fibers. Separation of tightly clustered fetalLeydig cells and expansion of intercellular spaces mayresult from a reduction in cell size after the first week oflife. The observed changes in the average cell sizedetermined stereologically were not statistically signifi-cant in the present material between ages 11 and 15days; however, Mendis-Handagama et al. (1987) ob-served a 50 % reduction in the cell size during the thirdweek of life. Perhaps this decrease begins earlier, atleast in some of the cells. Taken together, the reductionof cell size seems to be associated with a decline insteroid production of fetal cells during both the peri-natal and early juvenile periods (for classification ofsexual development, see Adams and Steiner, 1988).
Our observations of an increase in collagen betweenthe Leydig cells together with a simultaneous decreasein the cell size and steroid production suggests thataccumulation of collagen is related to regression ofLeydig cells in developing rats. The change in amountof interstitial collagen (Hatakeyama, 1965; Pelliniemi etal. 1980; Schulze, 1984) as well as in the basementmembranes of Leydig cells (Kuopio and Pelliniemi,1989; Kuopio et al. 19896,c) in physiological, exper-imental and pathological conditions is further evidencethat the extracellular matrix may be involved in regu-lation of Leydig cells.
Our results indicate that a second, prepubertal phase
220 T. Kuopio and others
of growth occurs after the initial regression of fetalLeydig cells in perinatal animals. This growth period isfollowed by another phase of regression during thesecond postnatal week, coinciding with the develop-ment of adult-type cells (Mendis-Handagama et al.1987). Instead of the commonly accepted biphasicpattern of Leydig cell growth in laboratory rodents (deKretser and Kerr, 1988), there are in fact three consecu-tive stages of Leydig cell development in rats: fetal(fetal cells in fetal testis), early juvenile (fetal cellsduring neonatal-early juvenile life) and juvenile-adult(adult cells before and after puberty). This patternresembles the situation in the pig, which no longer canbe considered an exception (Dierichs et al. 1973; deKretser and Kerr, 1988) among the species.
Supported by grants from the Sigrid Jus61ius Foundationand from the Academy of Finland.
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