THE JOURNAL OF EXPERIMENTAL ZOOLOGY 243:481-487 (1987)

Ontogeny-Related Changes in the Peptide Profiles of the Germinal Epithelium: Analysis by Sodium Dodecyl Sulfate Gradient Gel Electrophoresis

MEENAKUMARI AND S . DURAISWAMI Department of Zoology, University ofDelhi, Delhi-110 007, India

ABSTRACT The premise that one manifestation of the nexus between Sertoli cells and germ cells may be an orderly and sequential change in their protein profiles has been examined in relation to the ontogeny of spermatogen- esis in the colony-bred albino rat. Viable “Sertoli cell-germ cell associations”1 isolated from the testes of animals of defined postnatal age and incubated in an appropriate medium were separated into a Sertoli cell and a germ cell fraction and processed for analysis by sodium dodecyl sulfate gradient gel electrophoresis. The resulting stained bands were “mapped” and assigned relative mobility values by comparison with standard marker proteins. This enabled identification by serial number of individual bands from an overall total of 163. For purposes of detailed analysis, they were classified into high, medium-high, medium, and low molecular weight bands. Two major categories were delineated: 1) those associated uniquely with a specified day of ontogeny and 2) those appearing intermittently. Significantly enough, not one of the bands was encountered on all days examined. The relevance of the patterns observed to the possible exchange of “information” between Sertoli cells and germ cells during spermatogenesis is mooted.

It is generally recognized that the Sertoli cells play an indispensable role in mamma- lian spermatogenesis. Data from an autora- diographic study of the ontogeny-related protein synthetic capabilities of Sertoli cells on the one hand and germ cells on the other were taken as “strong evidence for some kind of interaction” between these two cell types (Meenakumari and Duraiswami, ’86a). What form could such interplay assume at the mo- lecular level? Developmental biologists con- sider that the process of differentiation is nothing but the programmed sequential-and differential-expression of genes during on- togeny (Maclean, ’77). As such, one would expect orderly and sequential changes in their protein profiles as one manifestation of the nexus between the Sertoli cells and the germ cells. Accordingly, we report here the results of a study, based on analysis by so- dium dodecyl sulfate gradient gel electropho- resis, of changes in the peptide profiles of germ cells and Sertoli cells during the tran- sition of the colony-bred albino rat from the sexually immature to the mature state.

MATERIALS AND METHODS Animals

The male rats used in this study were from our Holtzman-derived colony, maintained under standard animal house conditions. An- imals of postnatal day ages 8, 14, 18, 23, and 30 were used for this investigation. The rea- son for restricting the study to the above days of ontogeny has been presented else- where (Meenakumari and Duraiswami, ’86a), Only rats born on the same day were used for a given experiment. Six to 30 testes were employed depending on the selected age group-

Chemicals The standard molecular weight marker kit

(MW-SDS-70) consisting of bovine serum al-

’The term “Sertoli cell-germ cell associations” is used to designate the groups of Sertoli cells together with the associated germ cells (in other words, a part of seminiferous epitheliumj that are recovered after enzymatic removal of tunica propria from the seminiferous tubule fragments (Welsh and Wiebe, ’75; Meenakumari and Duraiswami, ’86b).

0 1987 ALAN R. LISS, INC.

482 MEENAKUMARI AND S. DURAISWAMI

bumin, egg albumin, pepsin, phenylmethyl- sulfonylfluoride (PMSF)-treated trypsinogen, 0-lactoglobulin, and lysozyme with molecu- lar weights (Mr) in kilodaltons (kD) of 66,45, 34.7, 24, 18.4, and 14.3, respectively, as well as sodium dodecyl sulfate (SDS), were pur- chased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. Tris (hydroxymethyl) ami- nomethane of analytical grade was bought from Fluka, Switzerland, and Coomassie Brilliant Blue R250 from Serva, Heidelberg. All other reagents were of analytical grade and were purchased locally.

Preparation of sample The Sertoli cell-germ cell associations

(SGAs) were isolated from testes of each spec- ified day of ontogeny by treating washed seminiferous tubule fragments with collage- nase and pancreatin (Meenakumari and Du- raiswami, '86b) and were incubated at 32°C for 60 minutes in a Dubnoff shaker water- bath. This was followed by conversion of the SGAs into single-cell suspension by manual dissociation, layering on an appropriate step- wise gradient of Ficoll-400, centrifugation, aspiration of the resulting discrete bands, identification of the cell type(s) contained therein, and finally, pooling appropriate frac- tions so as to furnish a Sertoli cell-enriched and a germ cell-enriched preparation for analysis. (For details of procedure, see Meen- akumari and Duraiswami, '86b.) The respec- tive samples were pelleted and resuspended in chilled polyacrylamide gel electrophoresis (4"C, PAGE) sample buffer (62.5 mM Tris- HCI, pH 6.8; Laemmli, '70) and the cells were disrupted by using a vortex mixer. The final volume was made up to 5 ml with the same buffer and 1 ml of chilled 50% trichloroacetic acid (TCA) was added. The samples were then stored in ice for 45 minutes. The precipitate was recovered and washed twice with chilled 5% TCA and, finally, given a rapid wash with ice-cold distilled water. Following resus- pension in the sample buffer, the precipitate was stored in the deep freeze until ready for gel electrophoresis.

Solid SDS to 2% and 2-mercaptoethanol to 5% were added to the TCA precipitate in sample buffer. The mixture was kept in boil- ing water for 2 minutes. The resulting clear solution was incubated overnight at 37"C, at the end of which period its volume was noted. Sucrose was added to an aliquot to give 40% concentration and this was subiected to

Vertical slab gel electrophoresis Polyacrylamide gel electrophoresis with SO-

dium dodecyl sulfate (SDS-PAGE) was car- ried out in an apparatus designed in our laboratory with dimensions of 12 x 15 x 0.2 cm according to the procedure of Laemmli ('70). The resolving gel was made of a continuous gradient of the acrylamide (7.5%-15%), whereas a 4% gel was used for stacking.

Appropriate volumes of samples were loaded in the wells. A mixture of standard SDS-marker proteins prepared in the same way as the samples was loaded in one of the wells. Electrophoresis was carried out at 4°C for 16 hours at constant current (20 mA per plate). Addition of bromophenol blue (track- ing dye) to the samples helped monitor prog- ress. At the end of the run, the gel was fixed overnight in 50% TCA, stained in 0.1% Coo- massie Brilliant Blue R250 in 50% TCA for 5-6 hours a t room temperature and de- stained with 7% acetic acid.

"Mapping" of the stained bands Mapping of the stained bands was per-

formed under transillumination of the gel placed on a transparent glass plate. The re- sulting map was carefully checked against prints of negatives obtained by photograph- ing the gels through an orange filter. The entire operation was carried out indepen- dently by three investigators. In all cases, there was complete concordance.

In order to facilitate comparisons, the total number of bands observed was divided into four groups as follows: a) high molecular weight bands (HMWB) with molecular weights greater than or equal to 66 kD (Mr 2 66 kD), b) medium-high molecular weight bands (MHMWB: 66 kD > Mr > 45 kD), c) medium molecular weight bands (MMWB: 45 kD > Mr > 24 kD), and d) low molecular weight bands (LMWB: Mr < 24 kD).

Relative mobility (Rm) values for the bands in each group were calculated by using the positions of the following markers as refer- ence (Rm=1.0): a) HMWB-bovine serum al- bumin (66 kD), b) MHMWB-egg albumin (45 kD), c) MMWB-trypsinogen (24 kD), and d) LMWB-lysozyme (14.3 kD). Some bands in the LMWB category moved ahead of the 14.3 kD marker and so have Rm values greater than 1.0.

RESULTS AND DISCUSSION

The distribution of the various cell tvDes in PAGE. the seminiferous epithelium for the sG&ified

ONTOGENETIC PEPTIDE PROFILE OF GERMINAL EPITHELIUM 483

However, this does not in any way vitiate the analysis of the electropherograms since these were examined only for the presence or ab- sence of specific bands. Nor does it affect the main thesis of the paper, viz., the appearance of a peptide, or a group of peptides, including subunits of multimeric proteins, as a func- tion of ontogeny.

As is evident from Figures 3-7, it is curious that not one of the 163 bands is present throughout all the age groups studied, whether in the Sertoli cell or the germ cell compartment. Such a finding, particularly in respect of the Sertoli cell fractions, is un-

Fig. 1. Typical SDS SLAB-PAGE electropherogram. This run was an analysis for the eighth day of ontogeny. Lanes 1 and 7: Sertoli cell-germ cell associations. 2 and 3: Germ cell fraction. 5 and 6: Sertoli cell fraction. 4: Markcr proteins (top to bottom) are bovine serum albu- min, egg albumin, pepsin, trypsinogen, @-lactoglublin, and lysozyme with Mr (in kD), respectively, of 66, 45, 34.7, 24, 18.4, and 14.3.

days of ontogeny is given elsewhere (Meen- akumari and Duraiswami, '86a). The method of analysis of the information furnished by the electropherograms has been detailed in the previous section. Figures 1 and 2 illus- trate the process for day 8. Based on single- dimensional SDS-PAGE, a total of 163 dis- tinct stained bands could be identified. In light of the technique used, it is recognized that a stained band with a defined Rm value may well represent two or more peptides with identical or near-identical molecular size.

10

.25 2 66,000

- - - - 3 1 4 , 3 0 0 - - - - - - Fig. 2. Typical peptide "map." This one is based on

the electropherogram shown in Figure 1. The method for obtaining accurate drawings from the stained gels is described in the text. The six lines at the extreme right mark the positions of the six Mr reference proteins. Depending on its location in relation to these markers, each band was assigned to one of four size categories, viz., HMWB, MHMWB, MMWB, and LMWB. Within each category, the bands were assigned relative mobility values, assuming relative mobility (Rm) = 1.0 for the particular reference protein. They were then serially ordered for each size category, pooling the data from all the days of ontogeny examined. The small lines to the immediate right of the Sertoli cell/germ cell column of bands draw attention to those present in Sertoli cell- germ cell association but missing from one or the other fraction. Stippling indicates a faintly stained band, dot- ted lines stand for more prominent staining, and contin- uous lines signify well-stained bands. MWB, molecular weight bands; H, high; MH, medium-high M, medium; L. low.

484 MEENAKUMARI AND S. UURAISWAMI

14 - 18 0 0 8 14

0 0 R u m

4 0 0 !slQlnl 5E

0 0 0

10 n

U l amu

45

25 0 0 m U

0 65 0

70 H!JD 0 0 EllnI 0

0 0

74 rrl

( C o n t d 1 Figs. 3-7. Scoring for bands of each size category

(HMWB, etc.) for designated day of ontogeny (indicated by the numbers on top of each column of rectangles). On the left margin, the serial number of every fifth band is indicated. Code: Empty rectangle-the designated band (example, HMWB-1 on day 8) is entirely absent on the given day of ontogeny. Left half filled (horizontal lines) but right half empty-band present in germ cell fraction

on!y. Left half empty but right half filled (vertical l i n e s t band present in Sertoli cell fraction only.

Figs. 3, 4. High molecular weight peptide bands (HMWB group) are those equal to or greater than bovine serum albumin (Mr = 66 kD) in size. The grand total of bands in this size category is 74.

usual if it is believed that proteins associated with “household functions” would be present throughout the life span of the Sertoli cell. The closest approximation to a universal oc- currence is exemplified by MHMWB-21 and LMWB-16, which are found in both the Ser- toli cell and the germ cell fractions of SGAs isolated on days 8, 14, 18, and 23. As a corol- lary, therefore, it appears that household functions, in the somatic cell compartment as much as in the germ cell compartment, are liable to change depending on the stage of ontogeny.

For the rest, the bands have been catego- rized as noted below, with a view to facilitat- ing analysis:

1) Those associated uniquely with a spec- ified day of ontogeny (category 1 bands) and

2) Those that appear intermittently, sometimes cyclically, during the ontogenetic period under study (category 2 bands).

Category 1 bands Bands belonging to this category excite

particular interest in light of the reasonable hypothesis that their presence, either as

ONTOGENETIC PEPTIDE PROFILE OF GERMINAL EPITHELIUM 485 8 14 18 23 30

n L_J

O U O C ~ ~ 2 8 0 Hruu D wmm 17mm

Fig. 5. Medium-high molecular weight peptide bands (MHMWB group). Mr less than bovine serum albumin, but equal to or greater than egg albumin (Mr : 45 kD). Grand total of MHMWB group bands is 28.

cause or consequence, must have special sig- nificance for the given stage of progression of spermatogenesis. Such a view receives sup- port from the observation that their numbers fluctuate in relation to day of ontogeny, day 14 registering a minimum with seven and day 18 a maximum with 22 bands. The other noteworthy feature is that while most are common to both Sertoli cells and germ cells, some are present exclusively in one or the other compartment.

The observations for day 8 are a case in point. It is interesting, to begin with, that even the relatively undifferentiated somatic and germ cells associated with this day of ontogeny are characterized by the presence of as many as 13 unique bands, ten of which are in the HMWB category. Of these, HMWB- 62 and MHWB-5 are found only in the Sertoli cell compartment, whereas HMWB-19 and - 36 are unique to the germ cell fraction. In light of the consideration that the Sertoli cell fraction is 96% pure and the germ cell frac- tion carries only a 1% contamination of Ser- toli cells (Meenakumari and Duraiswami, ’86b), it is highly unlikely that the bands shared by these two fractions are due to cross-

contamination. Presumably they represent “household proteins” of undifferentiated cells.

In contrast to the above, all the unique bands in the day 14 profile are shared by Sertoli cells and germ cells. While the Sertoli cell fraction was 92% pure, the germ cell fraction was contaminated with Sertoli cells to the extent of 23% (Meenakumari and Du- raiswami, ’86b). In this circumstance, it is hazardous to speculate on the significance of these shared, unique bands. On scoring the cell types present on day 14 (Meenakumari and Duraiswami, ’86a), it was observed that the two typically prominent germ cell types were the type A spermatogonia and the early spermatocytes. The bands unique to day 14, therefore, may be a correlate of the interac- tion between the germ cells and the Sertoli cells. It is of interest that the low number of unique bands (seven) seen on this day is as- sociated with a similar low (47) in the total number of bands in the profile.

Day 18 appears to be characterized by a high-in fact the maximum-number of unique (22) and total (70) bands. Among the unique bands, HMWB-22 and LMWB-4 are associated specifically with the Sertoli cell fraction, while HMWB-5, MHMWB-8, and LMWB-25 are present only in the germ cell fraction. The cell types encountered on this day are type B spermatogonia, resting as well as dividing, and, most prominently, mid- pachytene spermatocytes (Meenakumari and Duraiswami, ’86a). Inasmuch as the latter continue to be present in the germ cell frac- tion on day 23 and day 30 also, it appears reasonable to conclude that the unique germ cell bands of day 18 are associated with the type B spermatogonia which register a marked decline on days 23 and 30.

The 11 unique bands in the profile for day 23 are all common to the Sertoli cell-and germ cell-compartments and are obviously characteristic for the day of ontogeny. On this day, further differentiation of spermato- cytes (leptotene --+ zygotene -+ pachytene) is continuing, a process that will culminate in the first appearance of round spermatids on day 30 (Meenakumari and Duraiswami, ’86a).

The unique bands seen with the germ cell fraction (HMWB-25 and -63; LMWB-8) and the Sertoli cell fraction (MHMWB-27, MMWB-3) respectively on day 30 may be un- ambiguously ascribed to the presence on this day of the round spermatids. The postulate

MEENAKUMARI AND S. DURAISWAMI 486

Fig. 6. Medium molecular weight peptide bands (MMWB group). Mr less than egg albumin but equal to or greater than trypsinogen (Mr = 24 kD). Grand total of bands in this category is 32.

that the bands unique to the Sertoli cell com- partment, in particular, are round-spermatid related receives strong support from the au- toradiographic observation that the silver grains indicative of protein synthesis are characteristically localized in that part of the Sertoli cell cytoplasm that is adjacent to the round spermatid in the adluminal compart- ment (Meenakumari and Duraiswami, '86a).

Category 2 bands In this category belong those bands that

are present as components of the protein pro- files in Sertoli cells andor germ cells from more than one of the specified days of ontog- eny. Given that there is no single band found uniformly through all days of ontogeny stud- ied and also given that there are bands unique to a given day of ontogeny, what could be the significance of those that are shared by the cell populations belonging to two dif- ferent davs of ontogenv?

For instance, HMWB-15 and -33 are com- mon to days 8 and 14, while HMWB-61, MHMWB-22, MMWB-12 and -16, as well as LMWB-12 and -21 are shared by days 8 and 18. Band MHMWB-12 is common to days 8 and 23. Bands HMWB-38 and -47, MHMWB- 14, and MMWB-21 are common to days 8 and 30. All these shared bands are found associ- ated, in every instance, with both the Sertoli cells and the germ cells. The only common factor between day 8 and the other specified days is the presence in the germ cell com- partment of type A spermatogonia. Their number tends to decline with advance in on- togeny. On this basis and in light of the con- sideration that these shared bands are not common to all the stages under study, their presence cannot be attributed to the type A spermatogonia. The Sertoli cells, on the other hand, are common to all the age groups. On day 8, they are undifferentiated and are still dividing. A plausible explanation would be that in the undifferentiated state the Sertoli cells synthesize all of the above peptides, whereas their subsequent presence or ab-

- 0 nmm 0 0 0 U 0 D

30 0 0 €33

Fig. 7. Low molecular weight peptide bands (LMWB group). Mr less than trypsinogen. Grand total of LMWB

- . I group bands is 29.

ONTOGENETIC PEPTIDE PROFILE OF GERMINAL EPITHELIUM 487

sence is a consequence of differential gene expression.

An interesting variation on the above theme is that certain bands which at one stage of ontogeny are found in both Sertoli cell and germ cell fractions are later confined to either the one or the other compartment. For example, HMWB-12 is associated with days 14, 23, and 30. However, it is only on day 14 that it is found in both the compart- ments, whereas subsequently it is confined to the germ cell fraction only. Similarly, MMWB-4 is initially (day 8) associated with Sertoli cells as well as germ cells. On days 23 and 30, it is confined to the germ cell fraction. On the other hand, MHMWB-28 is present in both the compartments on days 14 and 23, whereas on day 30 i t is contined to the Sertoli cell fraction only.

The seeaingly total absence of a given band in a particular compartment may sig- nify nothing more than a fall in the level(s) of synthesis of the peptidds) composing that band such that its (their) presence could not be detected in the sample analyzed. Thus the cyclic appearance and disappearance of many of the shared bands, noted above, may be more a reflection of fluctuations in the over- all rates of synthesis of such peptides than a total cessation of synthesis. Regardless of the manner of interpretation of the “missing” bands, it is evident that there is a degree of cyclicity in the peptide profiles. Further ex- amples are LMWB-23 which is initially pres- ent in Sertoli cell and germ cell fractions on day 8, in Sertoli cell fraction only on day 14, in Sertoli cell and germ cell fractions on day 18, and missing on days 23 and 30, and MMWB-26, which is present in Sertoli cell fraction on day 8, missing on day 14, and present in both the fractions on days 18, 23, and 30. The existence of such patterns raises

the question of whether these bands repre- sent the so-called cyclic proteins described by Wright et al. (’83) in sexually mature rats. Even more dramatic is the case of band HMWB-66, which is an “early” protein in that it is first seen on day 8. It is missing on day 14 and when in reappears on day 18 it is found only in the Sertoli cell fraction. On day 23 it is present in both the fractions but on day 30 it is present only in the germ cell fraction.

Do such proteins as have been described above constitute “messages” from one com- partment to another during germ cell differentiation?

ACKNOWLEDGMENTS

This study received generous financial sup- port from the Indian Council of Medical Re- search, New Delhi. We thank Mr. E.A. Daniels for help with the photography.

LITERATURE CITED

Laemmli. U.K. (1970) Cleavage of structural oroteins during the assembly of the hYad of bacteriopdage T4. Nature. 227:680-685.

Maclean, N. (1977) The Differentiation of Cells. Edward Arnold, London, p. 34.

Meenakumari, and S. Duraiswami (1986a) Protein syn- thesis in the rat seminiferous epithelium during onset of spermatogenesis-an autoradiographic study. An- drologia, 18:474-482.

Meenakumari, and S. Duraiswami (1986b) A method for the isolation of intact Sertoli cell-germ cell associa- tions from rat seminiferous tubules and their further partition into Sertoli cell and germ cell fractions. J. Biosci. 10:413422.

Welsh, M.J., and J.P. Wicbe (1975) Rat Sertoli cells: A rapid method for obtaining viable cells. Endocrinology, 96:618-624.

Wright, W.W., M. Parvinen, N.A. Musto, G.4. Gunsalus, D.M. Phillips, J.P. Mather, and C.W. Bardin (1983) Identification of stage-specific proteins synthesized by rat seminiferous tubules. Biol. Reprod., 29:257-270.