Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats.

These studies examine the trophic effects of progesterone (P) on the progesterone receptor (Rp) and growth of the decidua basalis (DB) and junctional zone (JZ) in the rat placenta. Pregnant rats were ovariectomized (Ovx) in mid-pregnancy and received steroid replacement therapy consisting of implantation of P pellets (25 mg) and injections of estradiol (E), 2 micrograms s.c., daily. Placental protein synthesis, measured by 3H-leucine incorporation in vitro, decreased more than 99% within 24 h of Ovx. However, treatment with P immediately after castration maintained control levels of synthesis. Delay of P treatment for 4 h caused a 60% decline in protein production measured 20 h later (p less than 0.01). Intraperitoneal implantation of a 50-mg pellet of the antiprogestin, RU-38486, in intact pregnant rats decreased protein synthesis by 50% within 6 h and by more than 90% 12 h and 24 h post-implantation (p less than 0.01). Growth of DB and JZ in Ovx rats treated for 48 h with P and/or E was studied both histologically and by changes in protein and DNA content. Rp binding activity was also measured by exchange assay under equilibrium conditions. Only P was able to reverse the effects of Ovx on growth of the DB and JZ. P also maintained Rp levels in the DB above those observed in Ovx and Ovx + E-treated groups (p less than 0.01). The Rp may be a constitutive product in the JZ since binding activity was not altered by Ovx or by steroid treatments. This study shows that P is clearly a trophic hormone of the maternal and chorioallantoic placenta and is essential for placental growth, cellular differentiation, and histological integrity.

Structural correlates of stimulated and inhibited secretion: electron microscopic observations of somatotrophs in perifused rat pituitary.

The in vitro release of stored intracellular rat growth hormone (rGH) in response to several secretagogues suggests the functional division of rGH storage into at least two 'compartments' or 'pools'. The first is an immediately releasable compartment whose response is brief. The second is a compartment which responds to more prolonged secretory demands. These observations are consistent with either a single, homogeneous population of somatotrophs, each of which exhibits functional compartmentalization of storage, or with heterogeneous populations of somatotrophs, each family of which provides one of the observed responses. We sought anatomical correlates of this functional compartmentalization using a perifusion/morphometric system which permitted examination of the first model while not excluding the second model. We selected for study an established perifusion protocol whose behavior was consistent and whose previous results suggested phases of both filling and emptying of the immediate release pool. Five parallel perifusions of pituitary fragments were run. The fifth perifusion was used to monitor rGH release and to confirm that the experiment had behaved in standard fashion. The first pituitary chamber was dismantled during basal perifusion to obtain tissue for microscopy, the next during exposure to 25 nM SRIF, the third during exposure to both 25 nM SRIF and 1 mM (Bu)2cAMP, and the fourth shortly after the rapid release which followed SRIF withdrawal. Somatotroph granulation was decreased by 54% in the presence of SRIF, and then increased by 45% with the addition of (Bu)2cAMP. The intracellular distribution of granules also fluctuated in relation to the stimulatory and inhibitory secretagogues. In addition, secondary lysosomes increased by 549% during SRIF-induced inhibition of rGH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of estradiol and estradiol-progesterone treatment on gonadotropin secretion in castrate aging female rats.

Estradiol-17 beta and estradiol-17 beta-progesterone were administered to aging female rats immediately after castration. Estradiol (1.6 micrograms) alone suppressed the castration hypersecretion of FSH in mature animals to 70% of the castration controls, but in 360-day-old and older animals, the inhibition was less than 10%. There was a decrease in the castration hypersecretion of LH by exogenous estradiol-17 beta with increasing age, but the pattern of suppression was similar in all animals, regardless of age. When a constant dose of estradiol-17 beta of 0.4 micrograms/kg body wt. was administered to castrate rats with varying doses of progesterone, it was shown that after 270 days of age the negative feedback effect of these steroids on FSH secretion was not found. Whereas, when progesterone of varying doses was administered with a constant dose of 0.8 micrograms/kg BW of estradiol-17 beta, it was shown that after 180 days of age the positive feedback effect of these steroids on FSH was absent. After 270 days of age the augmentation of LH secretion by estradiol-17 beta and a middle dose of progesterone was not apparent even though the negative feedback on LH was effective. A defect in positive and negative feedback effects of gonadal steroids on gonadotropin secretion may be causative to age-related changes in the estrous cycle of rats.

Effect of ethanol-containing liquid diet upon gonadotropin receptor depletion in rat testis.

By using pair-feeding technique, the number of gonadotropin binding sites per unit mass of testicular homogenate was measured 15, 30, 45 and 60 days after ethanol administration to 60-day-old male rats. The ingestion of ethanol (5%, v/v) as a part of a nutritionally adequate liquid diet resulted in 30, 35 and 40% reduction in gonadotropin receptors by 15, 30 and 45 days, respectively. The body weights in the alcohol-fed rats were comparable to pair-fed controls. These findings suggest that the presence of ethanol in the liquid diet depresses the level of gonadotropin receptors in the rat testis.

Morphometric analysis of the autophagic and crinophagic lysosomal systems in mammotropes throughout the estrous cycle of the rat.

The crinophagic and autophagic lysosomal systems were studied in mammotropes (prolactin secreting cells) of the adenohypophysis throughout the estrous cycle of the rat. By means of morphometric analysis, it was found that the volume of secondary autophagic lysosomes was usually greater than that of the crinophagic type. Although the volumes of both secondary autophagic and crinophagic lysosomes were minimal throughout proestrus and diestrus 2, the autophagic lysosomal volume per mammotrope was elevated during the estrous period. The volume of secondary crinophagic lysosomes per mammotrope increased during late estrus and remained elevated throughout early diestrus 1. Furthermore, there was an inverse relationship between the volume of mature secretory granules per cell and of the crinophagic system. These data suggest a role for lysosomes in the regulation of synthesis and secretion of prolactin by the adenohypophysis of the rat.

Intracellular dynamics in pituitary mammotropes throughout the rat estrous cycle. II. Changes in synthetic and secretory organelles.

The organellar dynamics within mammotropes (prolactin secreting cells) of the anterior pituitary were examined using morphometry throughout the four-day rat estrous cycle. Two predominant periods of maximal organellar activity were found. The first period, which occurred during proestrus, resulted in two peaks of serum PRL at 6 pm and 12 m proestrus, while the second occurred during middiestrus-2 and resulted in an increase of pituitary PRL, but not a major serum PRL peak. The mitochondrial volume rose prior to that of other organelles during early proestrus, rising 110% from 4 am late diestrus-2 to 6 pm proestrus. However, the number of mitochondria per cell remained constant throughout the cycle. The volumes of rough endoplasmic reticulum (RER) and Golgi complex reached maximal proportions at 8 pm and 12 m proestrus, respectively. The RER volume was 153% greater than at 4 am late diestrus-2, while the Golgi complex had increased by 200%. We suggest that changes in the RER and Golgi complex volumes were the result of two processes: the synthesis of new membrane and the swelling of existing complexes. This study further substantiated that the serum peak of PRL at 6 pm proestrus is the result of stored mature granules being released, whereas the PRL peak at 12 m proestrus is due to the immediate release of hormone without any storage.

Intracellular dynamics in pituitary mammotropes throughout the rat estrous cycle. I. Morphometric methodology and hormonal correlations with cellular and nuclear volumes.

Serum prolactin (PRL) levels display regular patterns throughout the 4-day rat estrous cycle. A morphometric analysis of mammotropes (PRL-sereting cells) in the anterior pituitary was performed in an effort to quantify some of the ultrastructural events occurring prior to and during the secretion of PRL throughout the estrous cycle. In this study, a systematic sampling protocol applicable to the rat adenohypophysis was developed, which took into account the lack of synchrony in estrous cycles among the animals, the heterogeneity of the gland, and the number of cells required to yield a representative sample. These techniques were used to determine the cell, nuclear, nucleolar, and cytosol volumes, which were correlated with the changing levels of serum estradiol and PRL and pituitary PRL. Estradiol concentrations rose during late diestrus-1 and continued rising in a pulsatile manner, reaching maximal levels during proestrus. The elevations of estrogen were accompanied by increases in nucleolar and nuclear volumes. However, at early estrus and on the afternoon of diestrus-2, times at which it has been reported that progesterone levels are elevated, there was a dampening effect and these volumes declined. During early diestrus-2, when estradiol levels began to rise, there were increases in nucleolar, nuclear, cellular and cytosol volumes, as well as increases in pituitary PRL; but at late diestrus-2 there was a marked increase in nuclear and nucleolar volumes which was not accompanied by increases in cellular and cytosol volumes. During proestrus, two peaks of serum PRL were found, at 6 pm and 12 m, which were concomitant with increased nucleolar, nuclear and cellular volumes. The 6-pm proestrous peak of PRL was preceded by elevated pituitary PRL; however, the 12-m proestrous peak occurred when the pituitary PRL was minimal, even though nuclear and cellular volumes were elevated. This suggests that the 6-pm release of PRL was derived from previously synthesized and stored hormone, while the 12-m peak was the result of the immediate release of PRL, with little storage.

A computer program for the morphometric analysis of cell profiles.

A computer program which yields values for the volumes, surface areas, and volume/surface area ratios of cell profiles is described for use on a desktop calculator (minicomputer). This program uses standard morphometric procedures, and incorporates data obtained from electron micrographs at two levels of sampling. The main program yields values for the 'average cell volume' at the tissue level of sampling. Two options at the cellular level of sampling are also included which yield values for the volumes, surface areas and volume/surface area ratios for the organelles. The first option allows an analysis of 'whole cells' containing equatorial profiles through the nucleus, while the second option permits a 'fractional' approach using segments of the cells. Finally, some of the advantages of the two options are discussed.

Effects of aging on the hypothalamic-hypophyseal-gonadal axis in female rats.

The failure of reproductive function in aged rats could be due to deficiencies at the level of the ovary, pituitary, hypothalamus, or higher brain centers. The classic explanation that the ovary is depleted of follicles does not receive adequate support on the basis of histologic studies of aged ovaries. Basal serum gonadotropin levels change with increasing age in female rats. Serum follicle-stimulating hormone (FSH) levels rise while serum luteinizing hormone (LH) levels fall. Likewise, the characteristic response to castration is markedly altered in aged female rats with a reduced secretion of FSH and a minimal elevation of LH. However, the pituitaires of these animals are still caapable of responding to exogenous LH-releasing hormone with a delayed LH response whose magnitude simulates that seen in younger female animals. With increasing age there is decreased pituitary and/or hypothalamic sensitivity to the feedback action of estradiol. These data are consistent with the postulation that there is an altered hypothalamic-hypophyseal function in aged rats.

PIP: The hypothylamic-hypophyseal-gonadal axis in anovulatory retired breeder rats was evaluated to elucidate the changes occurring in aging female rats. Serum gonadotropins were measured and the response to castration and steroid replacement, ovarian responsiveness to exogenous gonadotropin stimulation, and pituitary responsiveness to luteinizing hormone-releasing hormone (LH-RH) determined. Castration and estradiol replacement in 22-month-old rats resulted in a reduced secretion of follicle stimulating hormone but at a lower rate than that seen in younger rats. The responses of serum LH to estradiol followed a similar pattern. Pregnant mare's serum gonadotropin given alone or combined with human chorionic gonadotropin in 22-month-old castrate rats caused a significant ovarian weight increase (p.01) over intact controls with a corresponding increase in uterine weight as well. Aging rats also responded to LH-RH (100 mg) with values of serum LH significantly elevated (p.01). These results suggest that the feedback mechanism of aging rats is less sensitive to stimulation than in younger rats. However, pituitaries are still capable of responding to stimulation. These data are consistent with the hypothesis of altered hypothalamic-hypophyseal function in aged rats.

Ultrastructural and physiological studies of the stalk-sectioned rat pituitary gland.

A pituitary stalk-section rat model was prepared that would be suitable for the study of the direct effects of steroids on the pituitary gland. In this model, the hypophyseal stalk was severed surgically and metal foil was inserted to prevent regeneration of portal vessels. The major portion of the pituitary no longer had a direct communication with the hypothalamus, and the only blood supply was from short portal vessels that supply the dorsolateral part of the gland. The effectiveness of the lesion was demonstrated by very high levels of serum prolactin and very low FSH and LH. Light microscopic examination revealed a large infarct in the central region of the gland with stainable cells in the peripheral area. Fourteen days after stalk-section, the infarct had shrunken appreciably, and after 21 days it was replaced by a scar. Ultrastructural studies revealed the presence of functional mammotropes in the peripheral regions of the pituitary gland, while other cells were smaller, less active, and contained fewer secretory granules. After LHRH administration there was an increase in serum LH, thus indicating the presence of a substantial number of viable gonadotropes. This was confirmed by the ultrastructural findings which now showed the appearance of large, well-granulated gonadotropes. When LHRH was given to estradiol 17-beta primed ovariectomized stalk-sectioned rats, there was a great augmentation of LH release, and many castration cells were now apparent. Therefore, the administration of estrogen and LHRH was needed for the gonadotropes of the isolated pituitary to function optimally in the synthesis and secretion of FSH and LH. The stalk-sectioned animal model permits evaluation of pituitary function isolated from hypothalamic control while it leaves the surviving pituitary gland in its normal anatomical position.

Actions of pregnant mare serum gonadotropin in the immature female rat: correlative changes in blood steroids, gonadotropins, and cytoplasmic estradiol receptors of the anterior pituitary and hypothalamus.

Several blood steroids, serum gonadotropins and cytosol estradiol receptors of the anterior pituitary and hypothalamus were quantified in immature female rats which were induced to ovulate with pregnant mare's serum gonadotropin (PMSG). Studies revealed that serum levels of progesterone, 17-hydroxyprogesterone, testosterone, androstenedione and estradiol were initially elevated at 6 PM (day 30) after administration of 8 IU of PMSG at 10 AM day 30. Serum levels of estradiol and testosterone rose progressively from day 30 through the AM of day 32. A further increase in serum concentrations of progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, and dehydroepiandrosterone occurred on the PM of day 32 whereas serum estradiol levels declined. Serum levels of all steroids declined on the day of estrus (33) and only progesterone levels were further elevated on day 34 (diestrus). Dihydrotestosterone concentrations were minimally altered by PMSG treatment. Saline administration resulted in no significant alterations in levels of any steroid quantified from day 29 to 34 in control animals. A progressive decline in cytosol estradiol receptor content of the anterior pituitary and hypothalamus was documented following PMSG treatment of intact female rats; there was no depletion of receptors following PMSG administration to ovariectomized immature rats. Maximal depletion of cytosol estradiol receptors occurred on day 32 with replenishment of cytosol estradiol receptor levels on estrus (day 33). The preovulatory gonadotropin surge was found to occur on the PM of day 32 after maximal receptor depletion. The cycle of depletion and replenishment of receptors was repeated during a second spontaneous estrous cycle four days later which coincided with a rise and fall in serum estradiol levels. It is suggested that the depletion of cytosol estradiol receptors of the anterior pituitary/hypothalamic unit may be causally related to the preovulatory gonadotropin surge resulting from PMSG administration to immature female rats. In addition, changes in blood steroids and gonadotropins after PMSG treatment are similar to those reported for proestrus-estrus-diestrus I of the normal adult estrous cycle. These findings further demonstrate the validity of the PMSG-primed immature female rat preparation as a model for the estrous cycle of the adult rat.

Influence of estrogen-progesterone combinations on gonadotropin secretion in castrate female rats(1).

In order to study the effect of progesterone in modulating the secretion of gonadotropins, various doses of progesterone were administered to castrated immature and mature female rats. Progesterone did not prevent the postcastration rise of FSH and LH in these animals. Various amounts of progesterone were then administered to castrated rats that were treated with a constant low dose of estradiol. In animals given doses of estradiol that were able to decrease serum FSH and LH below castrate levels, but not to prevent the post-castration rise, the effect of progesterone was dose-dependent. A very low dose of progesterone lowered serum gonadotropins, an intermediate dose brought about an increase in secretion, and high doses were suppressive. The modifying action of progesterone appears to be dependent upon the level of estrogen, the dose of progesterone, and the time of administration.

Dehydroepiandrosterone-induced polycystic ovaries and acyclicity in the rat.

Administration of dehydroepiandrosterone (DHA) to the immature female rat resulted in one precocious ovulation followed by ovulatory failure. The animals exhibited either "constant-estrus" or "constant-diestrus" vaginal smears, and the ovaries were either polycystic or contained corpus luteum-like structures. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels showed an ovulatory type surge on day 30, at the time of the precocious ovulation. Thereafter, serum FSH levels were comparable to those of control rats, whereas the LH levels were very low. Serum prolactin was elevated significantly in DHA-treated animals. The ovaries of DHA-treated animals were responsive to gonadotropins and the pituitary was responsive to luteinizing hormone-releasing hormone. Withdrawal of androgen treatment resulted in restoration of cyclicity. This animal model demonstrates that an androgen insult can cause ovulatory failure and polycystic ovaries in spite of normal ovaries, adrenals, and hypothalamic-pituitary mechanisms. That such ovulatory failure is a result of the androgen administration is further substantiated by restoration of cyclicity after androgen withdrawal. This animal model in several ways is similar to the human polycystic ovary syndrome and has a potential application in the study of the mechanism of androgen-induced ovulatory failure.

Primordial follicles with normal oocytes in the ovaries of postmenopausal women.

In ultrastructural studies, the ovaries of postmenopausal women were compared with those of pre- and perimenopausal women. Primordial follicles containing apparently normal oocytes were found in all postmenopausal ovary samples. Differentiating follicles were often found in these samples, but they were in various states of atresia. The findings suggest that the menopause does not result exclusively from exhausation of the primordial follicles.

Effects of various progestational preparations on gonadotropin secretion in ovariectomized immature female rats.

PIP: 26-day old female rats were ovariectomized and treated with 1 of 4 progestational steroids twice daily for 5 days. Follicle stimulating ho rmone (FSH) and luteinizing hormone (LH) levels were determined by radioimmunoassay to study the effect of these progesterones on FSH and LH. Progesterone and megestrol acetate did not significantly alter LH, FSH levels or change uterine weights. Norethindrone caused a significant increase (p less than .01) for all dosages; serum FSH levels were significantly lower than controls at the .03 mg/kg/day level (p less than .01) and serum LH was significantly higher (p .01) at the lowest doses given. At .3 mg dose, both LH and FSH were suppressed to intact levels. Medroxyprogesterone acetate (Provera) in doses greater than 2 mg/kg/day caused uterine stimulation (p less than .01) and suppre ssed FSH (p less than .01). At 8 mg/kg/day, serum FSH was still significantly higher than controls (p less than .01). Serum LH was suppressed compared with castrate controls at .5 mg (p less than .01) and was similar to intact controls at 2 mg. It is concluded that native progesterone cannot significantly lower gonadotropin levels in the absence of estrogens.^ieng