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Y ”..0-..0... 0..-.... ..0. . f. . .. .o. 0 .... o ... .00 o v . . . 40 ff..." 0". 90.0.u0, ..M‘k‘o .0 ABSTRACT CHANGES IN THE PARAVENTRICULAR NUCLEUS DURING DIFFERENT STAGES OF PREGNANCY AND LACTATION IN THE ALBINO RAT By Una Elizabeth Hutton The magnocellular cells of the paraventricular and supraoptic nuclei are known to synthesize the hormones oxytocin and yasopressin. Although much is known about the stimuli that release these hormones (uterine stimu- lation, mammary stimulation, and osmotic stimuli) much less is known about how the hormones are produced. The fact that cell sise and number of nucleoli in the cells of the supraoptic nucleus change during water deprivation and vasOpressin production led to the realisation that this phenomenon might also be true for cells in the para- ventricular nucleus during oxytocin production. The present study is an analysis of the paraventricular neurones in intact male and female rats as well as during pre- parturient, lactating, and post-parturient non-lactating states in the female. Una Elisabeth Hutton After histological preparation, the brains of four adult male rats, four adult female rats, four female rate one day prior to giving birth, four lactating mothers two days after giving birth, and four non-lactating females two days after giving birth were compared. Measurements were taken on percentage of cells containing two or more (multiple) nucleoli, percentage of cells whose nucleoli were bordering the nuclear membrane (marginated), and cell size. Both.medial and lateral cells of the paraventricular nucleus were observed and data for these cell groups were treated separately. Increases in the percentage of multiple nucleoli were seen for all three groups experiencing pregnancy conditions in comparison to the control females, and for the lactating females in comparison to males. For three of the groups studied, pre-parturient females, non-lactating females, and males, the lateral cells had a larger percentage of multiple nucleoli than the medial cells. Control hypo- thalamic cells showed no changes in percent of multiple nucleoli for any of the conditions studies. The ratio of percent multiples in the lateral cells to percent multiples in the medial cells was larger for males than females, and this was the only measure in which males differed from all the other females studied. Hargination proved to be a consistent variable to measure. No differences were found across groups or within Una Elizabeth Hutton animals when either one or more than one nucleolus was found within the nucleus. However, when the cell contained multiple nucleoli an extremely high percentage of these (97%) had at least one nucleolus bordering the nuclear membrane. Although cell sizes change during water deprivation and vasopressin production in the supraoptic nucleus, this phenomenon was not true for the paraventricular cells during increased exytocin production. However, a medial- lateral cell size difference was found. In the sagittal planes both these cell groups are approximately the same size. But in the horizontal planes the lateral cells are larger than the medial cells, and significantly larger than lateral cells in the sagittal planes. This is further evidence that the medial and lateral cells are two different cell populations. CHANGES IN THE PARAVENTRICULAR NUCLEUS DURING DIFFERENT STAGES OF PREGNANCY AND LACTATION IN THE ALBINO RAT B! Una Elizabeth Hutton A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1975 To The Other Una ii ACKNOWLEDGEMENTS I would like to thank Dr. Glenn I. Hatton for his continual support and patience throughout the formation of this thesis. I also appreciate the helpful comments and criticism.made by the other members of my committee, Dr. Lynwood Clemens, Dr. Edward H. Convey, and Dr. John 1. Johnson. Special thanks are offered to Mr. Villiam.Armstrong for the loan of his electric typewriter for so many hours. This research was supported by a NIH grant, # NS O9lh0, to Dr. Glenn I. Hatton, and to a Biomedical Sciences grant, # 71-088h, to the author. iii TABLE OF CONTENTS DEDICATION eeeeeeeoeeeeeeeeeeeeeeeeeeeeeeee ACKNOWLEDGEMENTS .......................... LIST OF TABLES eeeeeeeeeeeeeeeeeeeeeeeeeeee LIST OF FIGURES eeeeeeeeeeeeeeeeeeeeeeeeeee LIST OF APPENDICES eeeeeeeeeeeeeeeeeeeeeeee INTRODUCTION ..........................o... METHOD .................................... RESULTS ................................... DISCUSSION ................................ SUMMARY ................................... LIST 0? REFERENCES ........................ APPENDIX A (MATERIALS) .................... APPENDIX B (PROCEDURE) .................... APPENDIX C (RAw DATA) ..................... iv Page ii iii vi vii ll 15 35 53 55 62 6h 69 LIST OF TABLES Page Table 1. Summary of blood analysis data, number of pups, and time of perfusion of each 1.013310 animal eeoeeeeooeeeeeeeeeeeeeoe 16 2. Mean percent margination for medial and lateral cells of the para- ventricular nu010u3 eeeeeeeeeeoeooeeee 30 3. Cell areas of paraventricular neurones in um2 for all animals studied ....... 3h Figure l. 2. 3. A. B. C. D. LIST OF FIGURES Photomicrograph of a horizontal Thionin- stained section of the lateral cells of the paraventricular nucleus eeeeeeeeeeee Photomicrograph of a horizontal Thionin- stained section of the medial cells of the paraventricular nucleus ............ Photomicrograph of a horizontal Thionin- stained section of the lateral cells of thfi paraventricular DUOIOUB eeeeeeeeeeee Photomicrograph of a horizontal Thionin- stained section of the medial cells of the paraventricular nucleus ............ Mean percent (+S.E.) of multiple nucleol for medial, lateral, and non-para- ventricular hypothalamic control cells . Mean cell size in um2 for lateral and medial cells of the paraventricular nucleus .00...0.......OOOOOOOOOOOOOOOOOO vi Page 18 20 22 2h 1 27 33 APPENDIX A: APPENDIX B: APPENDIX C: LIST OF APPENDICES Materials eoeeeeeeeeeeeeeeeee Procedure eeeooeeeeeeeeeeeeee Raw Data eeoeeeeeeeeeeeeeeeee vii Page 62 6h 69 INTRODUCTION As early as 1906 it was discovered that posterior pituitary extracts had a stimulating effect on the uterus both in vivo and in vitro (Dale, 1906). Many years later it was suggested that milk ejection (ME) was a reflex which involved the activity of oxytocin (Ely and Peterson, 19141). Yet it was not until 1953 that the structure of oxytocin was analyzed by DuVingneaud and found to be a cyclic octapeptide linked by a disulfide bond. (CyS-Tyr-ILEU-Glu(MH2) - Asp(NH2) - CyS-Pro-LEU-Gly(MH2)) Since that time the work on this hormone has increased immensely with concentration on the areas of uterine motility, milk letdown, water regulation, and sperm trans- port, although the last is the least documented. Because of the well established effect of oxytocin on uterine contraction and milk letdown the female animal has been the primary subject of this research. The origin of oxytocin in the hypothalamus has been the subject of much debate. Although it was originally thought that oxytocin was secreted from the paraventricular nucleus (PVN) and vaSOpressin from the supraOptic l nucleus (SON), the evidence now supports the hypothesis that both hormones are formed in both nuclei, but are produced in separate neurones. The theory of the specialization of the PVM for oxytocin production arose from early lesion studies. Electrolytic lesions in this nucleus cause a marked reduction of oxytocin activity in the neurohypophysis (Olivocrona, 1957) and shuilar lesions cause dystocia in pregnant cats (Nibbelink, 1961). Both oxytocin and vasOpressin are found in the paraventricular nucleus and supraOptic nucleus (Lederis, 1961; Dyer et al, 1973). The more recent technique in which neurosecretory cells are identified by antidromic stimulation has given strong support for the production of oxytocin in both nuclei. A similar percentage of neurosecretory cells in both nuclei show a rapid acceleration in discharge rate about 15 seconds before a rise in intramammary pressure associated with ME (Wakerly and Lincoln, 1973a, 1973b). Other evidence for oxytocin production outside of the PVN is given by data showing that after bilateral electrolytic destruction of this nucleus in rats with diabetes insipidus, DI, rats lacking vasopressin, there are still substantial amounts of neurosecretory materials (probably oxytocin) stored in the pituitary one month later (Sokol, 1970). Studies linking the PVN with oxytocin production include those of stimulation and recording. Electrical stimulation of the PVN (Cross, 1955a; Cross, 1958a) and of the median eminence and neurohypophysis in the rabbit (Lincoln, 1971; Cross and Harris, 1950) resulted.in ME and/or induced labor. Recording studies of antidromically identified units in the PVN at different times of the estrus cycle have also shown interesting results. For example, the mean firing rates are higher during pro- estrus and estrus than in metestrus and diestrus. Firing rates at the end of pregnancy are twice that at mid- pregnancy. The firing rates at an hours after parturition and during lactation were higher than these during metestrus, diestrus, and earlier pregnancy states (Negoro et al, 1973). An increase in discharge rates can be correlated with hormone secretion (Dyball and Dyer, 1971; Brooks et a1, 1966). But interpretation of the increase in discharge rate for the level of the hormone in the blood is questionable, for it may reflect release into the pituitary, and storage, of oxytocin. Swaab and Jonkind (1970) investigated hormone production in the PVN and SON by analyzing the neuro- secretory activity of these nuclei during each stage of the estrus cycle, pregnancy, parturition, and lactation, and in the adult male. The Golgi-specific enzyme, thiamine diphosphate phoSphohydrolase (TPP-ase), was used for measuring neurosecretory activity. Their method was a semi-quantitive histochemical method which determines changes in enzyme distribution, not in enzyme activity. The SON and PVN reacted very similarly under these conditions. Enzymatic distribution was higher during both u parturition and lactation, lower during light induced persistent estrus, and even lower during day 21 of pregnancy. Enzyme distribution rates seen in males and in metestrus females were similar and the latter showed lower rates than did females in all the other conditions. The low enzymatic distribution during metestrus is snmilar to the low firing rates of PVN neurones seen at this time (Negoro et a1, 1973). The similar reaction of both nuclei (the PVN and SON) at these times cannot be explained without knowing the level of each.hormone, oxytocin and vasopressin, in these cells. During light induced persistent estrus the gonadotrophin hormone level changes and this may be influencing the high enzymatic distribution seen at this time. Because blood estrogen and.prolactin concentrations are high on the day of parturition, one cannot overlook the possibility that this may have an excitatory effect on PVN neurones, which.may be an explanation for the high TPP-ase distribution seen on this day. In ovariectomized rats, firing rates in the PVN increase when estrogen is administered (Negoro et al, 1973). There are two cystine-rich proteins in the hypothalamus, known as neurophysin A and neurOphysin B. Neurophysin A has been labelled as the carrier protein for vasopressin and neurOphysin B as the carrier protein for oxytocin (Dean et a1, 1968; Burford et al, 1971). To determine if the PVN synthesizes oxytocin and the SON synthesizes vasOpressin, radioactive cystine was injected into the two 5 nuclei separately and the neurophysin A/B ratio calculated. The ratio was .76 3 .03 with PVN injections and 1.15 3 .16 with SON injections. Because this oxytocin neurophysin was labelled more in the PVN, one may conclude that more oxytocin is produced in the PVN, assuming that the association of neurophysin B with oxytocin is valid (Burford et .1, 1972; Burford et .1, 19714). Rats with bilateral lesions in the PVN show a significantly larger neurophysin A/B ratio in the posterior pituitary when killed an hours after the cystine injections, further evidence for the majority of oxytocin production in the PVN (Burford et al, 1973: Burford et al, 197k). Although the manufacture of both oxytocin and vasOpressin may occur in two different nuclei, it appears that the individual neurones are specialized for production of only one hormone. In rats with hereditary diabetes insipidus and presumably incapable of synthesizing vaSOpressin, the neurohypophysis shows areas of clustered granules and other areas lacking in stainable material. In normal rats deprived of water, and secreting oxytocin, the granules are not clustered but are spread evenly throughout. When these normal animals are rehydrated, there is a reaccumulation of secretory granules with no segregated Clusters. These granules reflect the storage of oxytocin and vasopressin (Livingston, 1971). In the DI animals the non-staining areas may be the axon terminals of vasopressin producing cells (Sokol and Valtin, 1967) . Howe (1962) has shown that only certain areas of the neurohypophysis are labelled with arginine, an amino acid present in vasopressin but not oxytocin. In a later study, when twenty-one antidromically identified cells in the PVN were recorded during reflex milk ejection, there were nine cells which did not change their firing rates. These might possibly be cells responsible for vasOpressin production, or they may be inactive oxytocin producing cells. In our lib, with the light microscope, we have identified two different magnocellular cell groups in the PVN, the lateral and medial cell groups. It may be that one of these cell groups is responsible for oxytocin production, while the other is responsible for vasOpressin production. Moss, Urban, and Cross (1972) found that there are two sets of neurons in the PVN. Eighty-one percent of the antidromically identified cells were excited by acetylcholine and 83 percent were inhibited by norepinephrine. In the non- neurosecretory cells (those not identified by anti- dromic stimulation) 81% were excited by norepinephrine and 76% were inhibited by acetylcholine. However, this does not separate oxytocin producing cells from vasopressin producing cells. Further work on synaptic transmission is necessary for an understanding of how ADH and oxytocin are differentially released. Stimul for too Release There is a differential release of oxytocin and vasOpressin to different stimuli. Some stmmuli which are the most documented to cause release of oxytocin.are vaginal and nipple manipulation, while hemorrhage induces ADH release (McNeilly et a1., 1972) and .l M CaCl2 infusion releases both (Fersling et a1., 1973). ADH does have some milk ejecting activity although it is only 1/6 as potent as oxytocin (Cross and VanDyke, 1953). In many species, there is a rise in plasma levels of oxytocin associated with onset of suckling. Levels of 12—25 mU have been reported in the woman (Coch, 1968), l7-6ho mU in the cow (Folley and Mnaggs, 1965), and 31-375 mU in the rabbit (Bisset et a1., 1970). With a normal milk yield.in the rabbit, ADH was found in only one of eight animals (Bisset et a1., 1970). Measures of ADH have been taken in other animals during ME and have been found to be below the threshold necessary for ME. These animals include the cow (Petters and Coussens, 1950) dog (Pickford, 1960) and woman (Theobald, 1959). There is also favorable evidence for release of oxytocin during parturition, eSpecially during the second stage of labor; i.e., during the expulsion of the fetus. This has been reported in the sheep and cow (Fitzpatrick and Halmsley, 1965) goat (Folley and Knaggs, 1965) and in the rabbit (Haldar, 1970). In the rabbit it is apparent that parturition is a stimulus for release of oxytocin independent of vasOpressin. The ratio of oxytocin to ‘ vasopressin was as high as 26/1 in one case and at least 5/1. In the goat, neurophysin is released simultaneously with oxytocin, and no rise in ADH is seen (Meleilly et a1, 1972). Hakerly and Lincoln (1971) recorded.intramammary pressure in female rats with eight suckling young. The young were returned to their mother after 18 hours of separation. They found that there was a latency of 10-30 minutes after suckling began before there was a change in intramammary pressure. Thereafter there was an ME response every 10-20 minutes. This response is abolished by removal of the young, and is similar to that Obtained by a rapid injection of .5 -l.5 m0 of oxytocin. As was mentioned previously, there is a rapid acceleration in.firing rate in some of the PVN cells 15 seconds before this ME response (Lincoln et a1, 1973; Lincoln and‘Uakerly, 1971). It is interesting to note that suckling does not initiate re- lease of oxytocin. Although during ME the pups are seen to arch their back and pull harder on the nipple, this causes no further change in PVN activity. Because of the finding that bradykinin, 5-hydroxytrypta- mine, acetylcholine, and vasopressin will also cause ME, it had to be shown that oxytocin is the sole initiator of ME under normal conditions (Bisset et a1, 1970). N-carbamyl-quethyl, which competes for receptor sites with oxytocin, will block mammary contractions. But this drug is also an antagonist of vasopressin. Yet with the hydrated animals within 1.5 hours after surgery there is a normal urine output, with.an increase in urine flow following each.ME. This was further evidence that oxytocin is released independently of ADH during ME (Hakerly et a1, 1973). Nucleoli Although it is known that suckling of the nipple and stimulation of the uterus cause a rise in oxytocin secretion, the exact mechanism.by which cells in the PVN respond is not known. It has been reported that cells in the SON increase their number of nucleoli within two hours when rats are water deprived (Hatton and Halters, 1973). This increase continues to rise up to 2h.hours under deprivation conditions. With rehydration, the percent of multiples decreases although it does not return to the baseline level in 10 days of rehydration. Animals adapted to a desert habitat, where water conservation is necessary, are shown to have more multiple nucleoli in the supraoptic nucleus than animals adapted.te a more moist environment (Hatton et a1, 1972). The nucleolus is intimately concerned with the production of high.molecu1ar weight ribosomal RNA, and the production of proteins. Although there is a high concentration of protein within this nucleolus, it has not been determined if this organelle is a site for storage or synthesis of protein 10 (Busch and Smetana, 1970, p. 6). Yet the presence of multiple nucleoli may reveal one of the mechanisms at work in protein formation in these neurosecretory cells. The possibility that there is a nucleolar response of this kind during the increased production of the octapeptide, oxytocin, has now been analyzed. Statement of purpose The nucleoli of cells in the PVN have not been observed under conditions of lactation and parturition, except on day 8 of lactation where nucleolar size was measured. The presence of multiple nucleoli would be further evidence both of the role the PVN plays under these conditions, and of the underlying mechanisms at the cellular level for hormone secretion. The purpose of this eXperiment is to observe the number, position of nucleoli, and cell size in the PVN in the adult female rat, adult male rat, in the pre-parturient female, and in the post- parturient female under both lactating and non-lactating conditions. Although oxytocin is known to be produced in approximately equal amounts in both the normal male and female, the reason for the high presence of this hormone in the male remains unknown, and it will be interesting to compare the oxytocin producing cells of the two sexes. Because some preliminary work (Hatton and Hutton, unpublished) has shown that there are two populations of PVN cells, the medial and lateral cells, which react as ll heterogeneous bodies under water deprivation conditions, these two cell pepulations will be examined separately in this study. All data will be analyzed separately for the medial and lateral cells to determine if there are really two separate mechanisms at work in these differentiated cells. All control adult male and female rats were kept on a constant light cycle, as opposed to the other groups which will be kept on a light-dark cycle (1h hours light, 10 hours dark). The purpose of this is to make the female acyclic, a light induced estrous condition, and to avoid oxytocic fluctuations which may occur in the cycling animal. It has been shown that firing rates in PVN neurones are higher during proestrous and estrous than during metestrous and diestrous (Negoro et al, 1973). Although it has not been shown that plasma levels of oxytocin vary during the different stages of the female's cycle, I feel that if firing rates do change there might be a multiple nucleolar difference as well. Thus I chose to make the control females acyclic, similar to the lactating and pregnant animals. METHOD Animals The subjects in this study are adult male and female albino rats obtained from the Holtzman Company in Madison, 12 Hisconsin. These animals were housed individually with food and water present at all times. Ambient temperature ranged from 21-23 C. Five groups of rats were used in this study: four constant estrus females; four adult males; four females were sacrificed one day prior to expected parturition; eight females were sacrificed H8 hours after parturition, four of which were left with six lactating pups, and four of which had their pups removed at birth. Total number of pups and weight of each pup was recorded at birth for females sacrificed after parturition, or at time of perfusion for the pre- parturient (PP) group. All control adult males and females were kept under constant light conditions, whereas the remaining groups were maintained on light-dark cycles (1h hours light, 10 hours dark). The adult males and light-induced females were kept in our colony room for 30 days prior to perfusion. Histological procedure Animals were weighed and anesthetized with other. A two-cc blood sample was taken from the heart of all animals and plasma hematocrit and specific gravity determined. Animals were then perfused transcardially with a 0.9% saline solution, followed by a formalin solution of one part 37% formalin and nine parts 0.9% saline. The brains were removed from the skull, dehydrated in alcohol solutions and embedded in colloidin. 13 From each group of four animals, two brains were sliced in horizontal sections and two were sliced in sagittal sections. The brains were sliced at 22 um and stained with thionin. Cell characteristics Magnocellular cells of the paraventricular nucleus (PVN) of the hypothalamus were analyzed. These cells are larger than the parvocellular cells and have a dark stain- ing nucleolus and cytoplasm. The nucleus is round and clear. The cell area of both.media1 and lateral cells was measured for 60 medial and 60 lateral cells per animal. Samples Of fifteen cells were taken from each horizontal or sagittal section containing the PVN, half of which came from each side of the brain. Only cells with observable nucleoli were taken for cell size measurements. The cells were magnified 2,170 times and the outlines of the perikarya were traced. Planimeter measures were used for the area determinations. Nucleoli Any dark staining, approximately round spot of the apprOpriate size (2-5um) within the nucleus was considered to be a nucleolus. One hundred and fifty cells from.each side of the brain were sampled, making a total of 300 cells per animal. Of these half were lateral cells (cells in the more lateral, dorsal, and posterior regions) and half 1h were medial cells (cells in the more medial, ventral, and anterior regions of the nucleus). Both the position and number of nucleoli were observed. The two classifications of cells can be seen in Figure I. Control cells One hundred control cells from each animal were compared to PVN cells. These cells were taken from areas immediately surrounding the PVN. Weave Samples were taken on 25 cells from.each section. Either odd or even thionin stained sections were listed. This was 7-8 sections in the horizontally sliced brain and 17-21 sections in the sagittally sliced brains. From these, six horizontal sections and twelve sagittal sections were chosen for counting. From.these fifty cells were counted in the horizontal sections, 25 from each side of the brain, and 25 cells were counted from each parasagittally sliced section. A Hhipple-Hauser reticule was placed in one eyepiece of a Zeiss microscope. Under low power the PVN was brought into focus. Then under a higher power objective (h0x) one part of the nuclear region was placed under the reticule and decisions about which rows or columns of the reticule the sampled cells were to come from were made before the microscope was focused under this power. The part of the 15 nucleus to be counted, while keeping medial and lateral sections separate, was varied from.upper left, upper right, lower left, lower right, and center. RESULTS Physiological data The physiological data are given in Table I for all animals with the exception of males on whom no blood data was recorded. Plasma protein, hematocrit measures as percent plasma, number of pups, and time of perfusion are reported. Two one-way analysis of variances were done to analyze both plasma protein and hematocrit counts on all the females in the study. It appears that the three groups of females that underwent pregnancy had higher hematocrits at the time of perfusion. However the AOV was non- significant (£?3.09, P .10, ggs3/12). The differences for plasma protein counts were also non-significant (£s2.73, P .10, g£§3/11). This may be due to the small sample size measured. The number of pups varied from.2-l3 and there appears to be no relationship between this measure and the blood measures or the percentage of nucleoli in the paraventricular nucleus. Medial and lateral cells The paraventricular nucleus was photographed and these photomicrographs are seen in Figure I. Horizontally sliced l6 Table 1. Physiological Data Plasma Number Hematocrit Time of Animal Protein of Pups % Plasma Perfusion Normal Female 1 6.u5 - 5h.o 10:00 AM 2 6.50 - 55.0 10:30 AM 3 7.00 - 56.0 11:00 AM u 6.60 - 5h.0 11:30 AM Pre- Parturient 10 6.00 8 59.0 11:15 AM 11 6.30 12 61.0 10:30 AM 12 6.50 11 61.0 98hb AM lh 6.50 2 52.0 12:00 PM Lactating Females l 6.h0 6 58.0 11:15 AM 7 5090 9 bueo 8330 A.“ 5 6.u0 10 57.5 103h5 AM Pups Removed 2 6.30 6 60.0 113h5 AM 6 7.10 13 - 38h5 PM 8 6.70 5 60.0 2300 PM 13 7.h0 12 62.0 83h5 AM 17 Figure 1A. A photomicrograph of a horizontally sliced Thionin-staineo section through the lateral cells of the paraventricular nuc1eus from a post-parturient non- lactating mother rat. l8 .18... 3.4.... ...? .e QVJN ...?“W‘ or .... . o . 1 fl... o.“ D. fiwm‘flo‘ . a Figure 1B. 19 A photomicrograph of a horizontally sliced Thionin-stained section through the medial cells of the paraventricular nucleus from a post-parturient non- lactating mother rat. 'e . 1.. d-zoz _U om: " ._.<.._ I Om SBWdll'an % 28 between group comparisons. There was a reliable increase in multiples (P< .05) from normal females to the three groups of females experiencing pregnancy conditions. The lactating (L) females showed a higher percentage of multiples than males (P( .05). There were no other significant differences between groups. Medial vs. lateral cell differences within groups were significant at P<..001 for the non-lactating (NL) group, P< .005 for the pre-parturient (PP) group, and Pt’.001 for males. There were no significant medial- lateral differences in the other two groups. Because the Duncan's range test between group comparisons combines both medial and lateral cells, a Milcoxon rank sum was also done to compare the groups while keeping medial and lateral cell data separate. In the lateral cell group, differences were significant at P < .05 between normal females and lactating (L) females. In the medial cell group differences were significant (P (.05) between normal females and the L group, and between males and the L group. By a one-way analysis of variance, a significant difference was seen in control cells (non-paraventricular hypothalamic cells) vs. eXperimental group cells on per- cent of multiple nucleoli (Esl87.77, g; = 1/15, P< .001). A test for correlated measures was done across all animals, comparing the medial cell reaction to that of the lateral cells. r = .7. 29 An analysis, using the Mann-Whitney U test, was done to compare the ratio of percent multiples of lateral cells to medial cells between males and females. The ratio of lateral to medial cells is larger in males than in females (P < .05 one-tailed test). Marginated nucleoli When only one nucleolus was found within the nuclear membrane, and this nucleolus was touching the nuclear membrane, it was considered to be marginated. Hhen two or more nucleoli were seen within the nucleus, and one or more of these were bordering the nuclear membrane, it was considered to be a marginated multiple cell. Table 2 gives the means for percent margination for all lateral and medial cells for each group. A mixed design analysis showed that there were no significant differences across groups, between medial and lateral cells, and.no inter- action for both single and multiple marginated cells. These percents for singles were acquired by taking the total number of cells with one marginated nucleolus and dividing by the total number of cells with only one nucleolus in the nucleus. Similarly, the percents for multiples were acquired by taking the total number of cells with two or more nucleoli, one or more of which was marginated, and dividing by the total number of cells with two or more nucleoli. 30 Table 2. Mean percent margination for medial and lateral 0011's Animal group Single Multiple lateral medial lateral medial Normal females 35-h 3h.6 9hol 100.0 Normal males 35.6 33.0 98.8 97.3 Pre-parturient females hls7 37.9 9hs9 99.3 Lactating females 39.9 37.9 97.3 97.1 Non-lactating romIOC 38 o 1 3Se 2 9h. 5 gun 1 31 Cell Size Mean cell areas in square micro-meters and standard errors were calculated for each animal, with medial and lateral cell data kept separate. An analysis by a two factor mixed design comparing groups, and medial-lateral differences within groups, showed no significant differ- ences. However, when an analysis using a 2 x 2 design was done comparing medial-lateral cell sizes across each plane of section, there were found to be reliable differences between horizontal and sagittal planes (§=l9.89, P( .001, g; = 1/8), a medial-lateral cell size difference (§=8.90, P c .005, g; = 1/18), as well as an interaction ($821.7, P<‘.001, df 1/18). When an analysis using tytests for related measures was done to compare cell sizes within each plane of section, it appeared that in the horizontal plane lateral cells are significantly larger than medial cells (£36.1h, Pt .001, g; = 7, two-tailed), and in the sagittal planes there is no reliable difference between medial and lateral cell sizes. Figure 3 shows the means for medial and lateral cell sizes across groups in both the sagittal and horizontal planes. Table 3 gives the mean and standard error of cell areas for each animal in the study, maintaining a medial-lateral cell group separation. Also given here is the mean for all medial and lateral cells in each plane of section. An analysis using g-tests for independent samples was done to compare cell size differences across planes for medial and lateral Figure 3. Mean cell size in umz for lateral and medial cells in males (M), control females (F), pro-parturient females one day before parturition (PP), lactating females 48 hours after giving birth.(L), and non- lactating females #8 hours after giving birth (NL). Mean cell areas are given for both horizontally and sagittally sliced brains. 33 ISO m IZO o0 I90 I70 I50 .0