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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
1973
Plasma Progesterone Concentrations and Ovarian Histology in Prairie Deermice (Peromyscus maniculatus Bairdii) from Experimental Laboratory Populations
Barry Douglas Albertson College of William & Mary - Arts & Sciences
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Recommended Citation Albertson, Barry Douglas, "Plasma Progesterone Concentrations and Ovarian Histology in Prairie Deermice (Peromyscus maniculatus Bairdii) from Experimental Laboratory Populations" (1973). Dissertations, Theses, and Masters Projects. Paper 1539624808. https://dx.doi.org/doi:10.21220/s2-7nsc-4k85
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. PLASMA PPDGESTEPONE CDNCCNTPATIONS AND OVARIAN HISTOLOGY I ’ IN PRAIRIE DEERMICE (PE.RCITYSCUS MANIOJLATUS RAIRDII) FROM EXPERIMENTAL LABORATORY POPULATIONS
A Thesis Presented to The Faculty of the Department of Biology The College of William and Mary in Virginia
In Partial Fulfillment Of the Requirements for the Degree of Master of Arts
by Barry Douglas Albertson APPROVAL SHEET
This thesis is submitted in partial fulfillment
of the requirements for the degree of
Master of Arts
t (XATl-L, P. 0 iLiis X]Author
Approved, July, 1973
(S- v u . EricOik L. Bradley, Ph. D.
C. RicnardTTferriiah, Ph. ■W) D. fl&itjh (- f- Robert E. u. Black, Phi D. ACKNOWLEDGMENTS
The author would like to express his appreciation to Dr. Eric L. Bradley and Dr. C. Richard Terman under whose guidance this investigation was conducted. Their dedication, motivation and steadfast support was inexhaustable. The author also wishes to thank Dr, Robert E. L. Black for serving as a committee member, and for his critical reading of the manuscript. Special thanks must also be given to Mr. Glen Bean for his interest and skillful construction of technical apparatus used through out the project, and to Miss Libby Frazier, Miss Linda Teague, Mrs. Jewel Thomas, Mrs. Patricia Kipps and Mrs. Pamela Van Arsdale for their invaluable technical assistance.
iii TABLE OF CONTENTS
Page ACKNOWLEDGMENTS...... iii
LIST OF TABLES...... V LIST OF FIGURES...... vi ABSTRACT...... vii INTRODUCTION...... 2 MATERIALS AND METHODS...... 5 RESULTS...... 14 DISCUSSION...... 30 LIST OF APPENDICES...... 39 BIBLIOGRAPHY...... 45 VITA...... 49
iv LIST OF TABLES
Table Page
1. Comparison of plasma progesterone (ng/ml) in the estrus cycle of control deermice...... 15 2. Body weights (g) , ovary weights (mg) and plasma progesterone levels (ng/ml) in population and control female deermice...... 19 3. Plasma progesterone (ng/ml) of nulliparous population and selected stages of estrus of control deermice...... 23 4. Ovarian histology of control and nulliparous population females...... 25
v LIST OF FIGURES
Figure Page
1. Standard curve for progesterone obtained by competitive protein binding assay...... 12 2. Mean plasma progesterone concentrations (ng/ml) for control deermice in each stage of the estrus cycle...... 17 3. Mean body weights (g) , ovary weights (mg) and plasma progesterone concentrations (ng/ml) of control and nulliparous population deermice...... 21 4. Control deermouse ovary showing follicles (F) and corpora lutea (CL)...... 2 7 5. Control deermouse ovary showing mature follicle (F) , corpora lutea (CL) and atretic follicles (AF) .. 2 7 6. Ovary from nulliparous population deermouse showing a follicle (F) , corpora lutea (CL) and atretic follicles (AF)...... 29 7. Ovary frcm nulliparous population deermouse showing a mature follicle (F) and large numbers of atretic follicles (AF)...... 29 8. Frequency distribution of plasma progesterone concentrations of control and nulliparous population deermice...... 25
vi ABSTRACT
B o d y weights, ovary weights, plasma progesterone con centrations and ovarian histology were studied in repro- ductively inhibited females from asymptotic laboratory populations and throughout the estrus cycle of control nulliparous prairie deermice. Progesterone was measured by a competitive protein binding technique. Body and ovary weights of population animals were significantly lighter than those of controls. Plasma progesterone concentrations of the population females were not significantly different from those of the control females. The control females, however, were shown to undergo cyclic changes in plasma progesterone concentration that were related to their estrus cycle stage. Thus, -.the mean plasma progesterone concen tration for the nulliparous population females was signifi cantly above the proestrus value and below the metestrus value of control females, but was not significantly differ ent from values observed for estrus or diestrus in those controls. Control female ovaries showed significantly larger numbers of follicles and corpora lutea than nulli parous population females, however, nulliparous population females exhibited significantly more atretic follicles than controls. These data suggest that nulliparous popula tion females could be cycling with respect to their ovaries and plasma progesterone, and that progesterone could be in volved in the reproductive inhibition observed. However, more than one mechanism of progesterone inhibition is suggested, with the possibility that a non-progesterone mechanism may be involved.
vii PLASMA PRQGESTEIpNE CONCENTRATIONS AND OVARIAN HISTOLOGY
IN PRAIRIE DEERMICE (PERDMYSCUS MANICULATUS BAIRDII)
FROM EXPERIMENTAL LABORATORY POPULATIONS INTRODUCTION
Numerical studies of natural populations of deermice
(Peromyscus maniculatus) have shown that population out breaks of this species rarely occur (Christian, 1961;
Terman, 1965). The evidence available suggests that deer
mice may be more sensitive to density-dependent factors
limiting population increase, and may possess a more effi cient mechanism for controlling population growth than is
true of more violently fluctuating forms such as Lemmus,
Microtus and Mus (Terman, 196 6).
Studies of experimental laboratory populations of
prairie deermice (Peromyscus maniculatus bairdii) have
demonstrated that population growth was controlled under
conditions in which excess food, water and harborage were
available. Cessation of growth occurred at widely differ
ent levels in populations maintained under identical con
ditions of the physical environment, and was achieved by
one of two means: either failure of young to survive or
cessation of reproduction (Terman, 1965, 1969, 1973). These
data suggest a density related feedback mechanism of popu
lation control by curbing reproduction that might involve
the hypothalamic-hypophysial-gonadial axis (Christian, 1963;
Christian, 1964; Christian and Davis, 1964; Christian, Lloyd
2 3 and Davis, 1965; Terman, 1969).
Reproductive processes of female mammals depend on the cyclic secretion of hormones by the adenohypophysis and ovary. The rhythm of the hormonal secretion, however, is not intrinsic to the adenohypophysis or ovary, but is regu lated by specific central nervous system areas, especially the hypothalamus. The female reproductive cycle culminates in ovulation which is triggered by the release of ovulatory levels of luteining hormone (LH), and follicle stimulating hormone (FSH) from the adenohypophysis (Rothchild, 1960;
Flerko, 1966; Mahesh, 1971; Labhsetwar, 1972).
Follicle maturation is controlled in general by FSH, and ovulation and corpus luteum formation by LH (Diczfalusy,
1969). The formation and release of these gonadotropins are regulated by separate hypothalamic factors, FSH-releasing
factor (Igarashi and McCann, 1964; Folkers, 1973), and LH-
releasing factor (McCann, Taleisnik and Friedman, 1960) .
The secretion of these releasing factors is controlled by cues coming to the central nervous system from external
sources, and by internally circulating levels of hormones
(McCann and Dhariwal, 1966). Following the identification of progesterone as the
corpus luteum hormone (Allen and Meyer, 19 33; Allen and
Wintersteiner, 1934), it was found that purified proges
terone possessed ovulation suppressing properties (Dempsey,
19 37; Makepeace, Weinstein and Friedman, 1936; Selye, Browne and Collip, 1936; Phillips, 1937). Progesterone alone has relatively little effect on FSH, but it does suppress the pre-ovulatory surge of LH, whereas the combined administra tion of estrogen and progesterone in large enough doses depresses circulating levels of both FSH and LH (Diczfalusy,
1969).
Plasma progesterone concentrations have been reported
in a variety of mammals including the goat (Thornburn and
Schneider, 1972), sheep (Thornburn, Bassett and Smith, 1969)
the pig (Guthrie, Hendricks and Handlin, 1972), mare (Plotka
Witherspoon and Foley, 1972), dog (Christie, Bell, Horth and
Palmer, 1971), guinea pig (Feder, Resko and Goy, 1968),
hamster (Shaika, 1971), rat (Feder, Goy and Resko, 1967) and
human (Johansson, 1969). However, little data have been
published on plasma progesterone concentrations in mice.
The purpose of these experiments was to examine plasma
progesterone concentrations and ovarian histology in normal
ly cycling female deermice and in experimental population
nulliparous deermice to determine if progesterone is a
central factor in, or correlated with a neuroendocrine mechanism producing inhibition of reproduction in laboratory
populations of prairie deermice. MATERIALS AND METHODS
Population Animals
Experimental populations were founded by four pairs of prairie deermice from eight different litters (60-100 days of age) (Clark, 1938) with the female of each pair being pregnant. The first litter of each pregnant female was killed at 21 days after birth. The second, and successive litters, (fertilized in the population) remained as part of that population. Six of the ten populations used in this study had reached population asymptote (Terman, 1965), and had not had young born into the population for a minimum of
117 days. The four remaining populations had not reached asymptote when sacrificed, however, they included nulliparous daughters so that correlations could be made with asymptotic populations. The range in age at death of experimental nulliparous population females was 94 to 486 days.
Control Animals Adult female deermice were paired with non-sibling males of the same age (60 day minimum) in no-contact cages which allowed reception of visual, olfactory and auditory cues from the male, but prevented contact by means of a partition made of two layers of hardware cloth separated by
3/4 inch. Two groups of control females were used in the
5 6 study- Group I consisted of 35 females in which the phase of the estrus cycle was determined by daily vaginal lavage, and 50 females that were never examined by vaginal lavage.
Group II consisted of 50 females, all of which were examined for stage of the estrus cycle by vaginal lavage. All control females were housed in the no-contact cages with males for a minimum of 21 days before vaginal lavage was begun. The range of ages at death of group I controls was 130-338 days, and for group II controls was 10 8-135 days. All control females had been paired for at least 35 days before sacri ficing.
Both experimental and control animals were maintained under artificial lighting, programmed for approximately 12 hours of bright light (four 40 watt flourescent tubes, 0 730 to 1930), and 12 hours of dim light (four 15 watt incandescent bulbs). Room temperatures were kept within a range of 216-
30°C. Food and water were supplied ad libitum.
Blood and Organ Collection Animals were rapidly anesthetized with minimal ether inhalation. Blood was obtained in the early afternoon from control and experimental population females by venipuncture of the ascending vena cava at the level of the renal vein using a heparinized syringe. The total time for collection of a blood sample never exceeded 2 minutes. The samples were immediately centrifuged at 3000 rpm for 15 minutes.
The plasma was drawn off, and frozen at -20°C until assayed.
The whole animal was then placed in 10% formalin. Later, 7 ovaries were removed from the carcasses, cleaned of fat,
lightly blotted and weighed as paired organs to the nearest .01 milligram.
Materials 3 1 a, 2 a-H -Progesterone (Amersham/Searle) with specific
activity of 53 Ci/m mole was evaporated to dryness and di
luted to a concentration of 50 yCi/ml with absolute ethanol.
Non-radioactive progesterone (Schwarz/Mann) was dissolved to a concentration of 100 ug/ml with absolute ethanol. From this stock, working concentrations of 100 ng/ml and 10 ng/ml were prepared every two weeks using absolute ethanol. All
steroids were checked for purity by thin layer chroma
tography .
Precoated thin-layer plates (Silica Gel 60 F-254, E. M.
Laboratories, Inc.) were used. Prior to use all plates were washed by ascending chromatography with methanol twice and
then once with absolute diethyl ether.
Florisil, 60-100 mesh, (Matheson, Coleman and Bell) was
used as obtained, and stored at 80°C until use.
Petroleum ether (anhydrous, b.p. 30-60°C), ethyl ether
(anhydrous), absolute methanol, ethylene glycol and 1,4-
dioxane were obtained from J. T. Baker Chemical Company.
Ethyl ether and petroleum ether (38°-45° fraction) were
re-distilled in glass prior to use. Methanol was spectro- photometric quality, and not further purified. Ethylene
glycol and 1,4-dioxane were liquid scintillation counting
grade and used as obtained. Benzene (Mallenckrodt Chemical 8
Works) was re-distilled in glass at 80°C prior to use. Abso
lute ethyl alcohol, (USP, U.S. Industrial Chemical Co.) was used as obtained. All solvents were stored at room temper
ature. Liquid scintillation grade PPO and POPOP were ob tained from Sigma Chemical Co., and stored at -20°C until used.
Binding protein plasma was obtained by cardiac-puncture
from pregnant albino guinea pigs in the latter part of
gestation, and stored in 0.1 ml aliquots at -20°C.
G1assware
All glassware was soaked for 2 4 hours in concentrated
^SO^-Potassium dichromate, washed in hot soapy water, rinsed
in double distilled water five times and dried at 100°C prior •
to use.
Extraction and Isolation
The method employed in this study is a modification of
the methods of Strott and Lipsitt (1967), Tullner and Hopper
(1971) and Attal and Engels (1971). Approximately 2000 dpm of la, 2a-H^-Progesterone was added to 15 ml conical
centrifuge tubes and evaporated to dryness with air at room
temperature to serve as recovery estimations. Two hundred
yl of mouse plasma was then added to each tube. Samples were extracted twice with 5 volumes of petroleum ether for
1 minute on a vortex mixer. The petroleum ether layer was
drawn off using pre-washed disposable Pasteur pipets, placed
in clean conical centrifuge tubes and evaporated to dryness. 9
The dried residues were dissolved in methanol:ether (1:1)
and spotted on thin layer plates. The plates were developed in ether:benzene (2:1) by ascending chromatography. Non
radioactive progesterone was used as a marker on each plate.
After development, the progesterone marker spots were lo
cated with a short wavelength ultraviolet lamp, and an area of each sample lane corresponding to a distance of 1 cm.
above and below the center of the marker spot (total area= O 4cm^) was scrapped off. The loosened silica gel was sucked
into a disposable Pasteur pipet which had been tightly
plugged with a small wad of glass wool (previously washed with 3 ml ethyl ether). The samples were eluted with 3.0 ml
ethyl ether into 10x75 mm culture tubes and evaporated to
dryness. Samples and a silica gel blank were taken up in
1.0 ml absolute ethyl alcohol and 200 ul was transferred to
counting vials for recovery estimations. The remainder was
evaporated to dryness under air for the binding assay.
Competitive Protein Binding Assay
The binding assay was performed in a walk-in cold room
maintained at 3°C. One microcurie of la, 2a -H^-Progesterone
(4.72 x 10”^m mole) in ethanol was evaporated to dryness
and dissolved in 30.0 ml of 0.04 M. phosphate buffer (pH.
7.4). Fifteen microliters of guinea pig plasma were added
and mixed gently for 1 hour at room temperature. One milli
liter of this binding solution was added to all assay tubes.
The tubes were placed vertically in a horizontal shaker and incubated for 1.0 hour at a speed of 230 shakes per minute. 10
At the end of the incubation period 4 mg of Florisil was
added to each tube using a calibrated spoon. The tubes were shaken for an additional 30 minutes. Five hundred Pi
of the supernatant was transferred to counting vials and
10.0 ml of Bray's solution was added. The vials were counted
for 20 minutes in a Nuclear Chicago Series 724 Liquid
Scintillation Counter.
A standard curve was constructed by plotting duplicates of standard progesterone of zero, 1.0, 2.0, 5.0, and 10.0 ng.,
versus the dpm of progesterone bound to protein. Nanograms
of samples were interpolated directly from the curve. (Fig
ure 1)
Specificity
When progesterone determinations were made on plasma
from three normal male deermice, the mean concentration
observed was 0.1 + 0.1 (SEM) ng/ml plasma.
Blanks
Methanol:ether (1:1) with no progesterone was spotted
on one lane of a TLC plate and designated as blank. After
development, an area of this lane equal in area and adjacent
to the standard lanes was scrapped, eluted, recovery esti
mated and subjected to the binding assay. Displacement of
bound H^-Progesterone of these blanks was equal to that of
the standard curve zero ng value.
Histology
Ovaries from control deermice in selected stages of 11
Legend for Figure 1
Standard curve for progesterone obtained by comoetitive protein binding assay. Each point on the curve represents the mean of dunlicate determinations. Radioactivity (dpm * 10 ) 20 18 22 24 16 12 14 iue 1 Figure rg»«oe (ng) Proge»»«fone lO 13 12 13 estrus, and experimental population nulliparous females were embedded in parafin, serially sectioned at 10 microns and stained with hematoxalin and eosin. Total numbers of follicles of type 5a, or larger, (Petersen and Peters, 196 8), and corpora lutea were counted for both ovaries of individ ual deermice. Follicles were classified as atretic if ovoid or irregular in shape, and filled with a non-cellu- lar matrix. The number of atretic follicles in the middle most section of the middle and the middle + or - one row were counted and the mean number per section for paired ovaries from individual deermice being recorded. All ovarian elements were counted under 80x magnification.
Statistical Analysis
Comparisons of body weights, ovary weights and plasma progesterone concentrations were made using the means of the experimental populations because the individuals within each experimental population are not statistically indepen dent. The data were subjected to linear correlation co efficient analysis and Student's t Distribution. When significant differences (<.05) between the mean squares occurred, Cochran's approximation was used (Snedecor, 1956).
The 5% level of probability was regarded as significant in all cases. RESULTS
The Control Female Deermouse Estrus Cycle(Table 1, Figure 2)
The length of the estrus cycle in control females was calculated in 42 animals that had been followed through a minimum of two full cycles. The range of length of the deermouse cycle was observed to be three to seven days, with a mean of 4.7 + 0.15 (SEM) days.
Cyclic variations in plasma progesterone concentrations were observed in group I control females. However, the num ber of animals in specific phases of the estrus cycle were low. The group II controls were assembled to increase the number of control mice and to obtain more information on cyclic variations of progesterone observed in the group I controls. The group II control females likewise exhibited variations in progesterone concentrations with different stages of the estrus cycle. The progesterone concentrations for group I and II controls were then statistically compared for each stage of the estrus cycle. No significant differ ences between groups were observed (Appendix A) , and the data from the two control groups were, therefore, pooled by stage of the estrus cycle.
Significant differences in plasma progesterone con centrations were observed between all stages of the estrus
14 Comparison of Plasma Progesterone Concentrations (ng/ml) -H -P M-l 43 I—I w o a p u p -P o u 0 (U 0 p p o p e C p 0 o 0 o 0 c 0 -H -H *H p i— i—1 rH P rQ P o P ■a cu fd P o p p 6 >1 O O P IQ Cd p CP CD p o 0 fij CO p o e O 0 CD a CD c fd O o p CO P as • • rd a a 1 a -H P O H p 53 w us p P p P Q w s p +1 CO Oo o o CO e P CO CO p CO P C (d p CD CP p D i—1 ID O CO CD C P CD p 0 CD s 0 0 fd £ a a 1—1 1—1 p p O O o cx> t"- i p O o o o o o i— i r-" CM o 04 P — +1 CO 0 CO p o p P P V V V V V V V V V » • • • • * . • • • * 1 P LD 1— ) p P VO VO 1 o o o w + 1 p — p co p CO • • • 1 CM 1 MCM CM 1 M vo O ID VO o ID rH s p p -H —1 CO CO 0 £ 0 V V 0 V * « • • • « • •H CM Mo CM CM o 1 o 1 Q p +1 — — 0 0 p p V V . • . 1 1 cn P MO CM P H P O +t ID CM 0 e P p cd 0. 0 0 V • ♦ VO rH CM CT\ VO 03 P P +1 0 P £ O' CD g a • 15 16
Legend for Figure 2
The mean plasma progesterone concentrations for control deermice in each stage of the estrus cvcle. Each bar represents the mean of 15-22 observations, with the verticle lines indicating the standard error of the mean. Figure 18 cycle. A 103% increase (P<.01) in the mean progesterone concentration was observed from proestrus to estrus, and a
177% increase (P<.01) in mean progesterone concentration occurred from estrus to metestrus. However, mean pro gesterone concentrations decreased (P<.1) by 52% from metestrus to diestrus. Figure 2 illustrates the mean plasma progesterone concentrations for control deermice in specific stages of the estrus cycle.
Four control females failed to become vaginally perfo rate. The mean progesterone concentration of these imper forate females was significantly lower than those levels noted for all stages of the estrus cycle (Table 1).
Plasma progesterone concentrations were also obtained from six deermice in the latter stage of pregnancy. The mean progesterone concentration in these females was signif icantly larger than for any stage of the estrus cycle of control females (Table 1).
Comparisons of Control and Population Females
Body Weights (Table 2, Figure 3) Nulliparous deermice from experimental populations showed significantly lower (P<.001) mean body weights than control females. Moreover, mean body weights of founding females, and of all females excluding founders were signif icantly smaller than those of control females.
Ovary Weights (Table 2, Figure 3)
No significant differences were observed between the Body Weights (g), Ovary Weights (mg), and Plasma Progesterone (ng/ml) in Population and Control Deermice •iH i—i (it +J .5- -P fd 8 tn CD CO 8 G CD
•H* [K r—I n to cn O O rH i S 0) G —i i— i— i -»e * LO , 1— „— * ID r * * s CO LO * cn H* I— 1 'si* LO o\ CT\ o 00 (N cn rH cn LO co CO r- O — +1 +1 _■* ft ■ • 1
. 19 20
Legend for Figure 3
The mean body weights (g) , ovary weights (mg) and plasma pro gesterone concentrations (ng/ml) of control and nulliparous population deermice. Each control bar represents the mean of 90 determinations of individual deermice, and each population bar represents the average of 9 population means. The vertical lines indicate the standard error of the mean. Figure o r o o U 6u 3 S*3od DlUSOJd *536oJd JS O 3U u) 6 (\ |UI o tJK ( 6 ) * (Bui) " «>yt »" jrn ^ ^ (i|6|3jv\ (i|6|3jv\ h 6|3 ; «»/■ y; m ^ROfl o o X q a o j o ; - v?» ;- v?» /*, '-»■ □ a. o a 3 21 22 mean ovary weights of control females and population
founding females. However, the mean ovary weights of popu
lation nulliparous females were smaller (P<.001) than those of control females.
When ovary weights were compared with plasma proges
terone concentrations in control females, no significant
correlation was demonstrated. However, population nul
liparous females did show a significant positive correlation
(P<.01) between progesterone and ovary weights (Appendix B).
Plasma Progesterone (Table 2, Figure 3)
No significant differences in the mean progesterone concentrations were observed between group I control females
and any category of population females. It should also be noted that when group I females were combined with group II
and compared with population females with respect to proges terone concentrations, again no significant differences were
found (Appendix C).
Table 3 presents comparisons of plasma progesterone
concentrations in population nulliparous females and control
females in specific stages of the estrus cycle. Control
mice had significantly lower (P <. 05) mean concentrations of
progesterone in proestrus, and significantly higher (P<.025) mean concentrations in metestrus as compared with population
females. Mean progesterone concentrations were not signif icantly different between population females and control
females in estrus or diestrus. Plasma Progesterone (ng/ml) of Nulliparous Population and Selected Stages of Estrus of Control Deermice -H CO S a f S -H O G 0 05 O G rd fa rd P 01 6 0 fa H P -H i—1 -H fa2 rd P O01 O O fa >“• i—G I G 0 ^ G r—1 co CFi oo ro in CO rH fa H+1 + ~H e o 0) rtf 0 P I—i 00 o fa CM r- o LO 01 P G G V P P 01 P 0 01 0 • •
i— LO i—1 OLO VO s W P -H 01 G G P 0 0 • » i vo O CM CM CM LO o P P -H V 0 w 0 • • • M C M C CM vo ■H - r CM P 1-H 1 + Q g P 0 101 01 G 0 0 ♦ 0 • ■ rH 1 - 4 CM co P hH O S o P O P fd G fa . • • •H -H 1 - 4 4-1 n M H r % P! P P 2 p P 0 0 p 0 0 G dP rd U P G G P p O G O o o 0 P 0 0 fa fa 0 (d G 0 0 • -H •H ■H H i - i 1 - 4 i i - 4 3 T T5 P P p P — 2 — G fa rd O 0 0 G 0 rd P O G > G 0 O 0 0 0 0 rd 0 P P 0 0 G G £ G o • 1 1 23 24
Ovarian Histology (Table 4; Figures 4, 5, 6 and 7)
Population nulliparous females had significantly fewer follicles (P<.001), and corpora lutea (P <. 001) than controls. However, they averaged significantly more (P<.05) atretic follicles than control females. No correlation could be demonstrated between progesterone concentrations and numbers of follicles, corpora lutea or atretic follicles in control females or in population nulliparous females
(Appendix D). Ovarian Histology of Control and Nulliparous Females co CN "H -H ■H s I—I I s a +J 3 § O 03 co Pi rd rd
* -K ■fc * m O * CN CN CN 'sr cn o co oo IT) CN O'! CO 00 CN o un 3 0 -H 44 -H S S PP $*$ co r$*-$ • ■ rd
■H -H ■H S ‘d ° -S CO -H •rJ > cu T30) rH CN CO CN rH 3 U 03 U CP,® p P-.-H D > CD co Tj ro m m -P-M C PI o O id Q Q
O
*Q m - 4
25 26
Legend for Figure 4
Control deermouse ovary showing follicles (F) and corpora lutea (CL). X 64.
Legend for Figure 5
Control deermouse ovary shelving mature follicle (F) . Corpora
lutea (CL) and atretic follicles (AF). X 512. WwSSi
COLLEGE OF WILLIAM & MftftY 28
Legend for Figure 6
Ovary frcm nulliparous population deermouse showing follicles (F) r corpora lutea (CL) and atretic follicles (AF). X 64.
Legend for Figure 7
Ovary from nulliparous population deermouse showing a mature follicle (F) and large numbers of atretic follicles (AF) . X 320. 6
$jgp.g6E OF WILLIAM & MARX
WILLIAM & MftRY DISCUSSION
The data presented in this study have shown that circulating plasma progesterone concentrations in control female prairie deermice (Peromyscus maniculatus bairdii) fluctuate in a pattern similar to other spontaneously ovulating mammals. These fluctuations are, furthermore, reflected by cyclic changes in the vaginal epithelium during the estrus cycle. These data suggest that deermice exhibit a typical mammalian spontaneous biphasic ovarian cycle. It was observed that mean plasma progesterone con centrations increased from proestrus to estrus, (as ex pected in an ovarian follicular phase), reached their highest levels at metestrus, and then declined in diestrus,
(as would be observed in an ovarian luteal phase) (Figure 2). However, the maximum concentrations of progesterone observed in the deermouse at metestrus were higher than the majority of mammals previously reported; i.e., twice as much as that observed in the dog (Christie, Bell, Horth and Palmer, 19 71), three times that observed in the human female (Johansson,
1969), and approximately ten times that reported for the goat (Thornburn and Schneider, 1972) and mare (Plotka,
Witherspoon and Foley, 1972). Mean progesterone concentra tions in the deermouse most closely compared with those re-
30 ported for the golden hamster (Lukaszewska and Greenwald,
1969), perhaps reflecting the fact that both mammals are in the same subfamily, Cricetinae.
Four of the control females were vaginally imperforate, a condition suggested to indicate reproductive immaturity.
The mean progesterone concentrations measured in these fe males were approximately one half of the observed control proestrus values. Speculation about the physiological or endocrine condition of these imperforate control females must await further investigation.
Figure 3 illustrates the mean values for body weight and ovary weight of control and population nulliparous females. The fact that population females had significantly smaller mean body and ovary weights than control females suggests a possible inhibition of both total body develop ment and reproductive maturation.
We found a significantly positive correlation (P .01) between ovary weights and plasma progesterone concentrations in only the nulliparous population females (Appendix B) .
It has been established that progesterone concentrations can be correlated with numbers of corpora lutea in sheep
(Thornburn, Bassett and Smith, 1969). However, when ovarian elements, i.e. follicles, corpora lutea and atretic follicles were counted and compared with progesterone concentrations in control and nulliparous population deermice, no significant differences were found. Corpora lutea and follicles were ob served in all control females. However, certain population nul 32
liparous females showed no corpora lutea but exhibited high progesterone concentrations. In these cases it is suggested
that progesterone is being secreted from other sources.
Evidence has recently been presented that progesterone can be secreted by the adrenal cortex and uterus (Kind, 1971), and by the stroma or interstitium of the ovary (Lukaszewska
and Greenwald, 1969) . Further investigations into the nature
of the secretory products of the atretic follicles, that are
so characteristic of the inhibited animals, and the ovarian
stroma may prove to be important in understanding the in
hibited state. For example, if an aberrant ovary were pro
ducing excessive amounts of estrogen, there would be an
antagonism of the proper endometrial development which could
prevent implantation. We have not demonstrated any simple mechanism of in
hibition involving the chronically high level of proges
terone that could operate by inhibiting the secretion of LH
and thereby inhibit ovulation. It does, however, remain
possible that some of the population animals are inhibited
by just such a mechanism. Also, it is not necessary to
assume that only high levels of progesterone could effect
inhibition. For example, new evidence has been accumulated
which suggests that progesterone can exert both facilitatory
and inhibitory effects on mating behavior and time of
ovulation in the rat (Stevens, Spies, Hilliard and Sawyer,
1970), and in the golden hamster (Lisk, 1969; Reuter,
Ciaccio and Lisk, 1970). 33
It may also be that the animals are inhibited only by
degree and that all of the elements of reproductive poten
tial are present and they are simply operating at subnormal
levels or out of phase with respect to each other. Further, we must allow for the distinct possibility that other neuro endocrine mechanisms may be involved. A recent report by
Hixon and Armstrong (19 71) demonstrated that in the rat there
is an inhibition of gonadotropin-induced ovulation by pro lactin.
The variation observed in the plasma progesterone con
centrations among nulliparous population females could be explained by at least two very different processes. The
first of these is that most of the inhibited females would have shown a cyclic variation in plasma progesterone concen
tration if several plasma samples could have been taken from
them at various times. In support of this idea is the fact
that the range of progesterone concentrations overlap al
most exactly between population nulliparous and control fe
males and that 90% of the females of both groups exhibited
less than 45 ng of progesterone per ml of plasma, with the
largest number of females in both groups having plasma con
centrations between 5 and 15 ng/ml (Figure 8). Also, all of
the stages of follicular development were observed among
the various inhibited females, even though the mean ovarian
weight of nulliparous animals was smaller than the control
value. Moreover, 77% of the ovaries of nulliparous popula
tion females contained corpora lutea. If it is assumed 34
Legend for Figure 8
The frecruency distribution of plasma progesterone concentrations of control and nulliparous population deermice. Figure u f|outfuy f|outfuy jo jaqiur>N 35 36 that the presence of corpora lutea is evidence of ovula tion, then it is likely that FSH and LH were also being released in sufficient amounts to produce that ovulation
(Bingle 1969a, 1969b; Labhsetwar, 1972). Thus, these data could be interpreted to suggest that with respect to FSH and LH release, follicular maturation, and ovulation there may not be a total inhibition or turning off of reproductive function in nulliparous females.
A second alternative explanation of these data that cannot be ruled out with the evidence at hand is that the variance observed in ovarian histology and plasma proges terone concentration is not a result of cyclic changes with in individual animals, but rather that the animals are in different states of arrested reproductive function. Thus, it could be that the presence of corpora lutea in nulliparous population animals does not indicate ovulation within the previous four days but could be the result of either ovula tion occurring many weeks earlier or of the luteinization of unovulated follicles. Similarly, the progesterone values for individual animals could be chronically high, or low, rather than representing different points in a random sam ple of cycling animals. The additional observation that
87% of the nulliparous population animals were vaginally imperforate lends further support to the notion that, at least with respect to estrogen, these animals were not cycling. If this latter explanation is true then several states of inhibition may exist among nulliparous females 37 suggesting two or more stages of a single mechanism of inhibition or possibly more than a single mechanism at work. APPENDIX 39
LIST OF APPENDICES
Appendix Page
A. Comparison of estrus stages between Group I and Group I I ...... 40
B. Correlation of ovary weights with progesterone in population and control deermice...... ^1
C. Plasma progesterone concentrations (ng/ml) of experimental population and control female deermice (Group I and Group II)...... 42
D. Correlation of progesterone (ng/ml) with the number-.of follicles, corpora lutea and atretic follicles in control and nulliparous population deermice...... 40
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legend for Appendix D
Correlation (r) of plasma progesterone (ng/ml) with the number of follicles, corpora lutea and atretic follicles in control and nulliparous population deermice. APPENDIX D
Controls n r t P Progesterone with:
Follicles 20 0.078 0.335 n.s
Corpora Lutea 20 0.210 0.911 n.s
Atretic Follicles 20 0.144 0.617 n.s
Exp . I, Pop. 6A
Progesterone with:
Follicles 7 0.000 0.000 n.s
Corpora Lutea 7 0.564 1.529 n.s
Atretic Follicles 7 0.498 1.2 86 n.s
Exp. I , Pop. 1
Progesterone with:
Follicles 6 0.308 0.648 n.s
Corpora Lutea 6 0.743 2.220 <. 1
Atretic Follicles 6 0.184 0.348 n.s
Exp. II, Pop. 1
Progesterone with:
Follicles 10 0.048 0.136 n.s
Corpora Lutea 10 0.20 2 0.5 85 n.s
Atretic Follicles 10 0.544 1.833 n.s
Exp. II, Pop. 2
Progesterone with:
Follicles 7 0.245 0.669 n.s
Corpora Lutea 7 0.484 1.462 n.s
Atretic Follicles 6 0.46 8 1.059 n.s BIBLIOGRAPHY
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Barry Douglas Albertson
B o m on September 14, 1946, in Bloomsburg, Pennsylvania.
Graduated from Bloomsburg Area Junior-Senior High School in June,
1964. Received a B. S. degree in Biology from Juniata College,
Huntingdon, Pennsylvania. Began graduate studies at the College of William and Mary, Williamsburg, Virginia, in September, 1968.
Drafted by the U. S. Army September, 1969, serving on active duty until March, 1972. Returned to the College of William and Mary in June, 1972. Worked as a teaching assistant 1968 - 1969, and held a research fellowship 1972 - 1973. Currently a candidate for the degree of Master of Arts in Biology.