Long-term effects of neonatal ovariectomy on cerebellar development in the rat: a histological and morphometric study.

The effects of neonatal ovariectomy on cerebellar development and morphology were investigated in rats ovariectomized (OV) or sham-ovariectomized (SOV) 24 h after birth. At age 70 days rats were sacrificed and the cerebella processed for quantitative light microscopy of matched midline sagittal sections of the vermis. Body, brain and cerebellar weights, as well as total cerebellar area and area of the internal granular layer were significantly greater in the OV rats. The concentration (cells/mm2) and content (cells/section) of granule cells were significantly greater in each of the 10 vermal lobules of OV rats. The number of Purkinje (P) cells in the OV rats was significantly greater than control values for 6 lobules (L-III, L-V, L-VIb,c, L-VII, L-IX and L-X). P cell number for L-I, L-II and L-VIII was also higher in the OV group, but not significantly. The between group variation for P cell packing density (cells/mm) was significantly different, i.e. packing density for the OV rats was greater than the SOV value. The most consistent and significant difference between the folial patterns of SOV and OV groups was observed in L-VIII, the pyramis. A bilobed L-VIII occurred in 61.11% of SOV rats, but only in 16.67% of OV rats. Overall, neonatal ovariectomy significantly reduced the number of folia. Although the mechanisms are speculative, these data clearly indicate removal of the ovaries 24 h after birth, either directly and/or indirectly has long-term effects on cerebellar development.

Gender and gonadectomy influence specific proteins in the cerebellar cortex of the adult rat: a two-dimensional gel electrophoretic analysis.

Two-dimensional polyacrylamide gel electrophoresis was used to determine the influence of gender and gonadectomy on individual proteins of the 105,000 g supernatant (cytosol) of the cerebellar cortex of the adult rat. At age 60 days were gonadectomized or sham-gonadectomized. At age 140 days the rats were sacrificed and the 105,000 g supernatant prepared from the cerebellar cortex. Sham-operated female rats were sacrificed during the proestrus stage of the estrous cycle. Out of 200 proteins identified, 13 were influenced by gender and/or gonadectomy. Five of these proteins were influenced by both gender and gonadectomy. A total of 18 differences were observed. Apparent molecular weights and isoelectric points (pIs) for the 13 proteins ranged from 22,000 to 92,000 Da and pH 4.8-6.1, respectively. Sex differences occurred in the percent volumes (spot density) of 9 proteins between intact male and proestrous female rats. Sex differences in 4 proteins were not influenced by gonadectomy. These differences were presumed to be genetically programmed and/or to reflect the permanent organizational effects of gonadal steroids on the genome. Differences in the remaining 5 proteins depend upon the transient activational effects of the testicular sex steroids, i.e. these gender differences were eliminated by castration. The effects of gonadectomy on individual proteins were gender specific, i.e. proteins affected by ovariectomy were not influenced by castration and vice versa. This suggests intact gonads are required for the specific gender-dependent proteins identified in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Diminished quality of two-dimensional (2-D) gel electrophoresis after freezing of the first-dimension gels.

Two-dimensional (2-D) gels were compared before and after freezing mini isoelectric focusing (IEF) gels in their glass tubes at -20 degrees C. Overnight storage of mini IEF gels significantly reduced the separation, resolution and number of proteins in 2-D gels.

Cerebellar Na+,K+-ATPase activity is increased during early postnatal development of the estrogenized female rat.

The activities of Na+,K+-ATPase and Mg2+-ATPase were measured in the crude P2 synaptosomal fraction of the cerebellum through age 35 days in female rats injected s.c. with 500 micrograms estradiol benzoate 24 h after birth. Estrogenization did not affect Mg2+-ATPase. However, the activity of Na+,K+-ATPase was significantly increased above control values between ages 5 and 20 days. These data demonstrate an age-dependent estrogen-induced effect on cerebellar Na+,K+-ATPase during early postnatal development.

Effects of neonatal estrogen on hypothalamic deoxyribonucleases and DNA in the female rat.

Administration of a pharmacologic dose of 17 beta-estradiol benzoate to neonatal female rats during the sexually critical period of brain differentiation affected neither the activities nor developmental patterns of hypothalamic acid and alkaline deoxyribonucleases through 35 days of age. However, hypothalamic DNA content was significantly decreased in the treated rats at age 2 days. These data suggest that the transient steroid-induced decrease in DNA is neither mediated by acid nor alkaline deoxyribonucleases.

Splenic thymidine kinase and DNA during early postnatal development of the androgenized female rat.

Administration of a pharmacologic dose of testosterone propionate to neonatal female rats had no effect on either thymidine kinase activity or DNA synthesis in the spleen during the first 3 postnatal weeks.

Cerebellar deoxyribonucleases and DNA in the androgenized female rat.

Postnatal developmental patterns of activity for acid and alkaline deoxyribonuclease (DNase) were determined in the cerebellum of the androgenized female rat. Littermates were injected s.c. with either 1.25 mg testosterone propionate (TP) or vehicle 48 hours after birth. The enzymes and DNA content were measured in the cerebellum at 3, 6, 10, 15, 18, 22, 35, and 60 days of age. Neonatally administered TP affects neither the activities nor the developmental patterns of the DNases through age 60 days. However, the DNA content was significantly lower in the cerebellum of the TP-treated rats at ages 10 and 15 days. These data demonstrate that the TP-induced alterations in cerebellar DNA content are not mediated by acid or alkaline DNase.

Hypothalamic deoxyribonucleases and DNA content during development of the androgenized female rat.

The DNA content and activity of acid and alkaline deoxyribonucleases (DNases) were measured through age 60 days in the hypothalamus of female rats injected s.c. with 1.25 mg testosterone propionate 48 h after birth. DNA significantly increases through age 60 days, however, androgenization does not affect the DNases or DNA content. It is suggested that masculinization of the female hypothalamus does not involve alterations in net DNA synthesis and/or degradation.

Postnatal ontogeny of uridine kinase in the cerebellum, hypothalamus, and cerebral cortex of the rat.

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000 g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes in Km with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.

Ontogeny of thymidine kinase and DNA in the hypothalamus and cerebral cortex of the rat: effects of neonatal estrogen.

Developmental patterns of activity for thymidine kinase and DNA content were determined for the hypothalamus and cerebral cortex of the female rat during the first 3 postnatal weeks. The DNA content and thymidine kinase activity for both brain structures were maximal at ages 1 and 3 days, respectively. Total DNA content for the hypothalamus significantly increased 21% between ages 1 and 21 days. Administration of 17 beta-estradiol benzoate (EB) during the sexually critical period of brain differentiation had no effect on thymidine kinase in either brain structure. However, a transient and highly significant decrease in DNA content occurred in the hypothalamus of the 2-day-old EB-treated rat. These data indicate that (a) DNA synthesis occurs in the postnatal hypothalamus, (b) DNA synthesis in the hypothalamus and cortex is not tightly coupled to the activity of thymidine kinase and, (c) the EB-induced decrease in hypothalamic DNA is not related to changes in thymidine kinase.

The effects of sex and neonatal androgenization on thymidine kinase activity and DNA content in the cerebellum of the rat.

The effects of neonatally administered testosterone propionate (TP) were determined on the activity of thymidine kinase and on the content of DNA in the cerebellum of the rat during early postnatal development. Forty-eight hours after birth, female and male pups were injected s.c. with either 1.5 mg TP or an equivalent volume of vehicle. Cerebellar thymidine kinase activity and DNA content were measured at 3, 4, 6, 7, 8, 10, 12, 15, 18 and 21 days of age. A sexual dimorphism in thymidine kinase activity was found for control rats between 6 and 18 days of age, i.e. the activity of the enzyme was significantly higher in the cerebellum of the female controls. The activity of thymidine kinase in the cerebellum of TP-treated male rats was significantly greater than that of the male controls between 4 and 21 days of age. The activity of thymidine kinase in the cerebellum of TP-treated female pups was significantly lower than that of the female controls between 6 and 12 days of age. Enzyme activity at age 15 days was the same for TP-treated and control female rats. However, thymidine kinase activity in the cerebellum of TP-treated female rats was significantly increased above that of the controls at 18 and 21 days of age. Thus, the response of the enzyme following the injection of TP 48 h after birth was sex-dependent between 4 and 15 days of age. Alterations in cerebellar DNA content in the TP-treated male and female rats closely paralleled the changes described for thymidine kinase. These data suggest sex-dependent transactions between the animal and the injection of TP as well as androgen-dependent relationships between thymidine kinase and DNA synthesis in the cerebellum. Significant changes in the Michaelis-Menten constant with increasing age also suggest that the developing cerebellum contains more than one form of the enzyme.

Effects of neonatal estrogenization on thymidine kinase activity and DNA content in the cerebellum of the rat.

Cerebellar thymidine kinase activity and DNA content were determined following the administration of 17 beta-estradiol benzoate (EB) to neonatal rats during the sexually critical period of brain development. Male and female littermates were injected s.c. 24 and 72 h after birth with either 500 microgram EB or vehicle. At 3, 4, 6, 7, 8, 10, 12 and 15 days of age cerebellar thymidine kinase activity and DNA content were measured. A sexual dimorphism in thymidine kinase activity occurred in control rats between 6 and 15 days of age, i.e., enzyme activity was significantly higher in the cerebellum of the female rats. Thymidine kinase activity in both male and female EB-treated rats was (1) significantly greater than that of their littermate controls at 3 and 4 days of age, (2) the same as that for their controls at 6 days of age, (3) significantly lower than control levels between 7 and 12 days of age and, (4) the same as their controls by day 15. Alterations in cerebellar DNA content were similar to those described for the enzyme. These data demonstrate the occurrence of age-dependent estrogen-induced relationship(s) between thymidine kinase activity and DNA synthesis in the cerebellum of the rat during early postnatal development.

The effects of neonatal androgenization on the in vivo transport of alpha-aminoisobutyric acid into specific regions of the rat brain.

The purpose of the present study was to determine if the administration of testosterone propionate (TP) to neonatal rats is followed in vivo by alterations in the transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid (AIB), into specific regions of the brain. Forty-eigh hours after birth, male and female rats were injected s.c. with either 1,25 mg TP or an equivalent volume of vehicle. Five, 10 and 17 days after birth, control and TP-treated rats were decapitated at intervals of 2, 5, 60 and 300 min after the i.p. injection of 0.25 muCi [1-14C]alpha-aminoisobutyric acid/g body weight. Twelve brain regions, i.e., amygdala, cerebellum, corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pons, pyriform cortex and samples of serum were analyzed in terms of disint./min/mg tissue and as tissue/serum (T/S) ratios. At the end of 300 min there was a significant increase in the active transport of AIB in all brain regions of the 5-days-old TP-treated rats. Similarly, by 300 min, the active transport of AIB was significantly increased in all brain regions sxcept cerebellum and pons of the 10-day-old TP-treated rats. The administration of TP to neonatal rats did not alter the accumulation and/or active transport of AIB in brain regions of the 17-day-old rat at any of the tested intervals. These data indicate that (1) neonatally administered TP enhanced (either directly or indirectly) the transport and/or accumuation of AIB in specific brain regions of 5- and 10-day-old rats and (2) the effectiveness of the steroid decreased with the age of the rat.