Damon C. Herbert

Immunocytological localization of LH, FSH, TSH and their subunits in the pituitary of normal and anencephalic human fetuses

SummaryImmunostaining with antisera to oLH, hCG, hLH, pLHβ, hFSH, hFSHβ, hTSHα and bTSH was used to delineate the gonadotropic and thyrotropic cells of the human fetal anterior pituitary. Hypophyses from 29 normal fetuses, 3 newborn infants, and 5 totally ancencephalic fetuses were used. Several controls to check for the specificity of the immunocytological reaction were made.In normal fetuses, observations showed that: 1) the α subunit was detected from the eighth week and throughout gestation without sex differences; 2) intact LH was detected during the third month, however, age and sex differences were observed during the fourth and fifth months; 3) intact FSH was detected in female fetuses from the beginning of the fourth month, a sex difference was observed; 4) LH and FSH were detected in the same cells; 5) the thyrotropic cells were detectable from 15 weeks of gestation and their number increased during gestation without sex difference; 6) at birth the gonadotropic cells were scarce and were located in the ventromedian zone of the anterior pituitary, while the thyrotropic cells remained numerous and were located in the dorsomedian zone.In anencephalic fetuses: 1) the α subunit existed at each stage studied; 2) the reaction induced by anti-pLHβ and anti-hFSHβ sera was always very weak regardless of sex or age; 3) the thyrotropic cells were more numerous in comparison to the gonadotropic cells. These data are discussed in terms of the relationship of the hypophysiotropic hypothalamic factors to the appearance and evolution of the glycoprotein hormones and their subunits.

Spontaneous Mutagenesis Is Enhanced in Apex Heterozygous Mice

ABSTRACT Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex+/−) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex +/− lacI + mice compared to frequencies from Apex +/+ lacI + littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex +/− lacI + mice were significantly elevated twofold compared to levels for 9-month-old Apex +/+ lacI + control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

Folliculo‐stellate cells and intercellular communication within the rat anterior pituitary gland

Folliculo‐stellate (FS) cell are agranular and arranged around a follicle. They contain the S‐100 protein and β‐adrenergic receptors. It has been suggested that they can act as stem cells, since they show mitotic figures, and could transform into granular or chromophilic cells according to the concept of a “cell renewal system.” Cell‐to‐cell interactions among pituitary cells have been described, and recent progress with freeze‐fracture electron microscopy has provided novel observations of the cell surface and gap junctions within the rat or teleost fish pituitary gland, or in cultured rat pituitary cells. In adult rats, the anterior pituitary was composed of lobules incompletely separated by a basement membrane. Follicles consisted exclusively of FS cells. Gap junctions were observed only between adjacent FS cells, in rare cases on the tips of their cytoplasmic processes. Thus, the FS cells, connected by gap junctions, made up a dense cellular network throughout the pituitary. Gap and tight junctions were absent on granular cells. Elongated follicles with columnar FS cells were observed in 10‐day‐old rats and were separated into smaller units. The number of gap junctions rapidly increased with age until 40–45 days of age. Few S‐100 protein positive cells were observed on day 10, along the marginal cell layer and near the so‐called postero‐lateral wing. The frequency of positive cells increased with age and by day 40; numerous cells were observed throughout the anterior lobe.

Effects of estrogen-induced hyperprolactinemia on endocrine and sexual functions in adult male rats

Chronic estrogen treatment can lead to development of prolactin (PRL) secreting pituitary tumors. We have tested the ability of diethylstilbestrol (DES) to produce persistent hyperprolactinemia (hyperPRL) in adult male rats and examined the effects of this treatment on hypothalamic-pituitary-testicular function, adenohypophyseal structure, copulatory behavior and fertility. Silastic capsules containing approximately 5 mg DES were subcutaneously implanted into adult male CDF (F-344)/CrlBR rats and removed 15 or 20 weeks later. Extreme hyperPRL, as well as suppression of plasma LH and FSH levels, persisted after DES capsules were removed. In contrast, plasma testosterone levels increased rapidly after removal of DES capsules and reached normal levels within 4-6 weeks. Copulatory behavior was assessed on two occasions between 7 and 14 weeks after removal of the DES capsules and was found to be suppressed in DES-treated rats, as evidenced by significant increases in latencies to mount, to intromit and to ejaculate. Moreover, when the animals were placed with normal females, the interval until conception was significantly greater in DES-treated than in control males. In spite of these differences in copulatory behavior, 10 of 11 DES-treated males were fertile. At autopsy, 44 weeks after capsule implantation (i.e. 24 or 29 weeks after capsule removal), DES-treated rats had marked enlargement of the anterior pituitary, increased weights of the lateral prostate and the adrenals, increased levels of testicular hCG-binding sites, reduced concentration of dopamine and norepinephrine in the median eminence and increased concentration of LHRH in the preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical study of the post-natal development of the folliculo-stellate cells in the rat anterior pituitary gland

We investigated the post-natal development of cell-to-cell communication within the rat anterior pituitary gland cells using immunohistochemistry of the S-100 protein. Tissues of animals from 10 to 60 days of age were analyzed. At 10 days of age, S-100 protein-containing cells were rarely observed. With age, the population of S-100 immunostained cells increased until day 40 when they were found to be quite numerous. No further changes were noted from day 40 through day 60. From our previous studies, we conclude that the cells which reacted with the S-100 antiserum were folliculo-stellate cells and their developmental pattern parallels that of the hypophyseal-gonadal axis.

TISSUE SPECIFIC EXPRESSION OF MOUSE TRANSFERRIN DURING DEVELOPMENT AND AGING

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

Morphological changes in prolactin cells of male rats after testosterone administration.

Intact and castrated 25- and 60-day-old male rats were given 200 mug of testosterone propionate (TP) daily for 15 consecutive days. The morphology of the prolactin (PRL) cells was examined by electron microscopy and analyzed by the point counting method of Weibel and Bolender (23). The PRL cells in the controls had long cytoplasmic processes that coursed between adjacent parenchymal cells. The Golgi complex was relatively small in these cells and the area occupied by the secretory granules was, in general, less than in the TP-treated animals. After TP administration, the cells became more ovoid and the Golgi complex enlarged, displaying dilated cisternae and many immature secretory granules. In addition, the diameter of the secretory granules was greater in the animals given the steroid. When the morphology of the PRL cells in the sexually immature control rats was compared to that observed in the older, sexually mature rats, relatively few differences were observed indicating that the response to TP was essentially the same in animals of both age groups. The concentration of PRL was measured in the serum and pituitary gland by radioimmunoassay. There was a significant elevation in PRL in both the serum and pituitary glands of all animals given TP. In contrast, castration led to a fall in PRL levels. From these observations and from the morphological data it can be concluded that TP is capable of stimulating the rate of PRL synthesis and release, and that testosterone may have a regulatory role in PRL biosynthesis and turnover in the male rat.

Fertility onset, spermatogenesis, and pubertal development in male rats: effect of graded underfeeding

ABSTRACT: Undernutrition has proven to be a useful model for exploring the relationship between growth and pubertal development in female rats, such as the “critical body weight” hypothesis of pubertal timing, but corresponding studies in the male have been hampered by lack of specific discrete markers of puberty similar to vaginal opening or first estrus in females. In the current study, we explored the effect of five different levels of food intake (as low as one-thrid of normal) beginning at weaning on pubertal development and timing in male rats, using the date of the initial successful conception with normal females as a discrete marker for puberty in males. In underfed males, there was a weak inverse correlation (r=–0.31, P<0.05) between the age at puberty and the growth rate, the latter being used as an index of the degree of underfeeding. In contrast, there was a strong direct correlation (r=0.78, P<0.001) between body weight at puberty and growth rate. In the most severely underfed groups, the Lee index of body fat remained subnormal before and after puberty. Initial litter size also tended to be reduced when the males were underfed. At age 51 days (prior to puberty), graded underfeeding led to progressive reductions in serum luteinizing hormone and follicle-stimulating hormone levels as well as in parameters of androgen status (serum and testicular testosterone, prostate, and seminal vesicle weights). Testicular size was also reduced, but daily sperm production rate was not greatly affected by underfeeding. Testicular histology at age 51 days revealed mature step 19 spermatids in all groups, with some reduction in seminiferous tubular diameter in the most severely underfed group. The effect of underfeeding on testicular testosterone and spermatogenesis appeared to decrease as underfeeding was continued for an additional 70 days. We conclude that graded underfeeding beginning at weaning leads to major reductions in gonadotropins and androgens but has less effect on spermatogenesis or the timing of puberty. Therefore, in normal rats, the timing of puberty is closely related to spermatogenic development, which can proceed once initiated despite low gonadotropin and androgen levels. Underfed male rats have low body weights and low body fats at puberty, refuting the “critical body weight” and “critical body fat” hypotheses of pubertal timing.

Influence of Protein-Calorie Malnutrition on the Circadian Rhythm of Pineal Melatonin in the Rat

Abstract Pineal melatonin was measured over a 24-hr period in 50-day-old Sprague-Dawley male rats that had been fed a low-protein diet for 30 days and in a group of age-matched controls that were maintained for the same period on a standard laboratory chow. The patterns of melatonin secretion were similar in both groups. The highest content and concentrations were recorded during the dark phase of the 1ight:dark cycle, while the lowest were measured after the onset of light. At each of the time intervals studied, less of the antigonadotrophin was present within the pineal glands of the malnourished rats.