Pattern of follicular development during the estrous cycle of aged rats

Summary The pattern of follicular development during the estrous cycles of aged rats was examined and compared with that of mature rats. In both, preovulatory follicles are derived from a select group of small pre-Graafian follicles which begin to develop at estrus and reach the preovulatory size by the morning of proestrus, but the rate of growth, as judged by an increase in the percentage of granulosa cells incorporating ³H-thymidine, is accelerated in the follicles of aged rats. A second mechanism, which accounts for preovulatory follicles in aged rats, involves the rescue from atresia of pre-Graafian and preovulatory follicles. The existence of this mechanism is supported by the observation that at metestrus in aged rats virtually all follicles, regardless of their state of atresia, possess a high percentage of granulosa cells incorporating ³H-thymidine, indicating that the follicles are growing rapidly. However, some of these rapidly growing follicles show signs of atresia such as pyknotic nuclei within their granulosa cell layers. Since follicles in the initial stage of atresia contain defective oocytes (Peluso et al. 1979b), their rescue and development into preovulatory follicles would result in the ovulation of defective oocytes, a fact which accounts in part of the lower fertility in these older animals.     

Role of estrogen and androgen in maintaining the preovulatory follicle

Summary The effects of Nitromifene citrate (CI 628), an antiestrogen, and Flutamide, an antiandrogen, on the ultrastructure and viability of the preovulatory follicle and granulosa cells were examined both in vivo and in vitro. In vivo administration of either antihormone induced degeneration within the granulosa cells. In some of the affected granulosa cells, the nuclear material was condensed while the cytoplasm and associated organelles were unaltered. In others, the density of the cytoplasm was reduced, the smooth endoplasmic reticulum was dilated but the nucleus remained unaltered. In vitro, either antihormone reduced granulosa-cell viability but the granulosa cells were twenty times more sensitive to CI 628 than to Flutamide. In addition, exposure to CI 628 induced nuclear condensation without affecting the cytoplasm, while Flutamide induced the deterioration of the cytoplasm without altering the nucleus. These observations suggest that: (1) both estrogen and androgens control the viability of the granulosa cells and thereby the follicle, (2) the action of estrogen and androgen is mediated through receptors within the granulosa cells since these antihormones prevent the nuclear uptake of their respective hormone, (3) the granulosa cells of preovulatory follicles appear to be more dependent on estrogen than on androgen, and (4) each steroid appears to have a specific role in maintaining the granulosa cell; estrogens control the integrity of the nucleus while androgens preserve the cytoplasmic organization of the granulosa cell.The authors are indebted to Dr. Neri of Schering AG for donating the Flutamide and to Dr. Westland of Warner-Lambert/Parke-Davis for providing CI-628     

Bovine ovarian follicular cysts: in vitro effects of lecirelin, a GnRH analogue

This study investigates the mechanisms of action by which a GnRH analogue may modulate the contractility of the bovine ovarian follicular wall. The in vitro evaluation of the spontaneous basal contractility of bovine preovulatory and cystic follicles was performed, followed by testing the effects of lecirelin, a GnRH analogue, on their basal contractility. Strips of tissue in isolated organ bath were employed.In addition, to better investigate the mechanism of action of lecirelin, the study of the effects of cumulative doses of nifedipine (a calcium channel blocker), phentolamine (an α-adrenoceptor antagonist) and reserpine (an inhibitor of the vesicular up-take of catecholamines) alone and, at the highest doses employed, associated to lecirelin, was set up.The results demonstrate that in basal conditions and after the addition of lecirelin, the strips from preovulatory follicles contract significantly more than strips from cysts.Furthermore, among the patterns of contractility evoked by the three drugs employed, the one induced by nifedipine was the only one unaffected by the addition of lecirelin.The data obtained provide the hypothesis that one of the main mechanisms of action of GnRH, could involve calcium channels.     

Morphological investigations on follicular atresia in canine ovaries

Summary Morphologically, canine ovaries show two types of regression pattern in secondary follicles. In type A necrotic changes of the oocytes and the zona pellucida dominate, whereas in type B degeneration, necrobiosis and necrosis of the granulosa prevail. The atretic course of type B regression results in a pseudoantrum and leads, by pseudogrowth, to a structure imitating tertiary follicles. In both type A and type B regression, four consecutive stages of atresia are distinguished by light- and electron microscopy.True tertiary follicles display only one regression pattern which resembles type B of secondary follicles. Early, advanced, late, and terminal stages of atresia are again described.     

Aspects of cellular proliferation during follicular atresia in the dog ovary

Summary Autoradiography after pulse labelling with [³H] thymidine was applied to investigate the proliferation processes in the granulosa and theca related to follicular atresia of the dog ovary during metestrus.The number of proliferating cells depends on the follicle type and its atretic stage. There is less proliferation in smaller secondary follicles than either in larger ones or tertiary follicles. While in early atresia tertiary follicles show the highest labelling indices, in advanced atresia the larger secondary follicles are those with the highest values. For each follicle type a decline in the labelling indices can be observed from early to terminal atresia. Tertiary follicles show a precipitous decrease in the labelling index between early and advanced atresia. There is a continuous gradient of proliferation from the center of the follicle over the peripheral granulosa to the theca. In tertiary follicles, an inverse correlation between labelling and necrosis of granulosa cells can be observed.     

Ultrastructural alterations in the initial segments and in the recurrent collateral terminals of Purkinje cells following axotomy

Summary Transection of Purkinje cell axons in adult male rats made 1.5 mm or further from the cell body does not lead to the death of the neuron and results in compensatory structural alterations of the surviving axonal portions of the nerve cell. Near to, and at the emergence of recurrent collaterals of Purkinje cell axons, huge varicosities filled with filaments, granular material, lysosomes and mitochondria develop. Terminals of recurrent axon collaterals also exhibit different degrees of structural changes. Most striking of the morphological alterations is the regular presence of nematosomes in the hypertrophic axonal branches, especially in synaptic terminals. Since nematosomes were shown to contain RNA in other types of neurons, their presence in recurrent collaterals may indicate an enhanced synthetic activity in Purkinje axonal processes and endings after axotomy.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

Structural changes occurring during atresia in sheep ovarian follicles

Summary The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques.In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present.During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 m but occasionally reached as much as 400 m. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus.In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular.Changes in the thecal microcirculation may play a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses.The authors are greatly indebted to Dr. H.M. Dott and Mr. G.C. Foster for carrying out the analysis with the Quantimet image analysing computer. The skilled technical assistance of Mrs. Linda Collins is also gratefully acknowledged     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.     

Ultrastructural observations on the postnatal development of the rat prostate

Summary The postnatal development of the rat prostate has been studied with the electron microscope. Major developmental changes begin during the second week after birth and involve organelles associated with the formation of secretions. The amount of granular endoplasmic reticulum and the size of the Golgi complex increase greatly. Large vacuoles that probably contain secretory material are formed, and the lumen of the prostatic acini appears to contain secreted material. Large lysosomes with polymorphic interiors are present as early as 10 days after birth, and they become numerous by the end of the third week. Differences in fine structure between the different lobes of the prostate are detectable in 10–14 day old rats. The subsequent differentiation of the granular endoplasmic reticulum into the forms characteristic of the different prostatic lobes is described. The initial changes in the prostate occur in advance of sexual maturity of the animal, and the adult appearance of the gland is attained by 4–5 weeks after birth.This study was supported by Contract No. 69-2104, Program Project HD-02282, Health Sciences Advancement Award FR-02084, and a Research Career Development Award (1-K3-GM-28, 214-03) from the National Institutes of Health.The author wishes to acknowledge the technical assistance of Mrs. Stephanie Krah.     

The ovarian follicle as a model for the cell-mediated control of tissue growth

Summary Thy-1⁺ cells, producing Thy-1⁺ material, have been demonstrated by the indirect immunoperoxidase technique in the theca of growing ovarian follicles of the rat. OX-2 antigen, known as the minor glycoprotein of rat thymocytes, was detected in granulosa cells of non-growing follicles. Ia⁺ cells of dendritic type and/or activated macrophages were identified in the granulosa of advanced degenerating follicles, and remnants of the zona pellucida exhibited immunoglobulins. In some ovaries immunoglobulins were also bound to the zona pellucida of oocytes of early degenerating antral follicles. Medium-sized antral follicles with degenerating granulosa were occasionally invaded by cells carrying antigens of cytotoxic T lymphocytes or other T lymphocyte subsets, while degenerating large antral follicles were sometimes invaded by cells exhibiting antigen of cells with natural killer function (but not antigens of T lymphocytes). Granulosa cells of some degenerating antral follicles exhibited class-I antigens derived from the major histocompatibility complex. We suggest that cell-mediated control mechanisms of antigen expression and metabolism of tissue cells during their differentiation and degeneration should be considered in addition to the well-documented hormonal dependence of some tissues.     

Ultrastructural study on the hypothalamic-hypophysial-adrenal axis in fetal rats

Summary The adrenal glands of decapitated and encephalectomized fetal rats were investigated electron microscopically and compared to those of normal intact fetal rats. Although the adrenal cortices did not show three zones (zona glomerulosa, fasciculata, and reticularis) on the 16.5th day of gestation when the decapitation or encephalectomy was carried out in utero, the zonation was recognized in fetuses operated on the 21.5th day of gestation. The same was true for normal control fetuses. However, cytoplasmic characteristics suggesting steroidogenesis in the cortical cells were reduced to various degrees in the encephalectomized or decapitated fetuses, especially in the latter ones. The change in cytoplasmic appearance was more conspicuous in the inner portion of the cortex. This result suggests that for the maintenance of normal adrenocortical function the hypothalamus may be indispensable even during the prenatal life of rats.     

Ultrastructural changes in the theca interna during follicular atresia in sheep

Sixteen antral follicles, 1.8--4.2 mm in diameter, at various stages of atresia, were studied by electron microscopy. Deletion of theca interna cells by condensation followed by fragmentation (apoptosis), with subsequent engulfment of the fragments by remaining thecal cells, was present at all stages, but was most marked during secondary and tertiary atresia. In primary and secondary atresia, the relative numbers of thecal cells whose cytoplasm was rich in tubular endoplasmic reticulum were higher than in non-atretic follicles of comparable size. During tertiary atresia the number of cell layers in the theca interna was reduced, and cells rich in tubular endoplasmic reticulum became proportionately less numerous. Degenerating cellular material was present within the lumina of thecal capillaries at all stages of atresia. Such material was rarely seen in primary atresia, and increased in incidence progressively in later stages. It was concluded that during atresia a large number of theca interna cells are deleted by apoptosis, and many thecal capillaries become blocked with cellular debris.     

Ultrastructural alterations in ciliated protozoa under heavy metal exposure

Transmission electron microscopy was used to study the ultrastructural changes induced by exposure to Cd or Zn in three species of ciliated protozoa: Colpoda steinii, Cyrtolophosis elongata and Drepanomonas revoluta. The main cytoplasmic alterations were partial mitochondrial degeneration, cytoplasmic vacuolisation, accumulation of membranous debris and autophagosome formation. At the nuclear level we detected nucleolar fusion in the macronucleus, and micronuclear membrane modifications. We compared these modifications with those coinciding with ciliate encystment (a differentiation process induced by environmental nutritional stress) and with changes in eukaryotic cells treated with staurosporine, a potent protein kinase inhibitor considered to be an apoptosis inducer. Exposure to heavy metals also coincided with the appearance of electron-dense accumulations in the cytoplasm, which might be related to metallothionein-mediated detoxification. The results are compared with previously reported data from ciliates and microalgae treated with heavy metals.     

Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus

Summary This report concerns a light and electron microscopic investigation of the median eminence and dorsal infundibular stem of the rat following surgical isolation (deafferentation). Using a modification of the Halász technique, the basal hypothalamus, including the arcuate nucleus and median eminence were surgically isolated from surrounding structures. Special attention was directed to the contact (external) zone of the median eminence and rostral infundibulum where tuberohypophyseal axons as well as ependymal cell processes abut upon the abluminal basement membrane of the portal perivascular space. The results of this study to date suggest that 9, 20, and 40 days following surgical isolation, there is a distinct increase in the population of tuberohypophyseal dense core vesicles. It is suggested that deafferentation abolishes inhibitory and excitatory input that serves to modify the cellular dynamics of tuberohypophyseal neurosecretory elements. Comments are also made on the presence of cistern-like structures in the lateral median eminence; the presence of vesicle-like inclusions in terminal ependymal processes is discussed in relationship to the role that ependyma may play in linking the third ventricle with the adenohypophysis.This research was supported by USPHS Grants NB 08171 and AM 10002. The authors are indebted to the excellent technical assistance provided by G. Krobisch Dudley. Further, the authors wish to express their thanks to Dr. Adolph Weindl for his valuable advice and criticism, and to Matilde Holzwarth for her helpful assistance.     

Ultrastructural study of neurovascular contacts in the median eminence of the rat

Summary In the median eminence of the rat axons of the supraoptic-paraventricular-hypophyseal tract with elementary neurosecretory granules (150–200 m) traverse the internal zone. Terminals containing dense core vesicles 60–120 m in diameter end on the portal capillaries of the median eminence. A unique organisation of the primary portal capillaries is shown. Endothelial cells have many fenestrae. The pericapillary space has numerous extensions all of which represent a special zone around the endothelial tube. The fine structural organisation and function of the neurovascular contacts in the median eminence are discussed.     

Complex interrelationships among CL,preovulatory follicle,number of follicular waves,and right or left ovaries in heifers

The diameter of the dominant follicle (DF) of wave 1 was studied on Days 9 to 17 (Day 0 = ovulation) in a survey of the ipsilateral and contralateral relationships between the location of the DF and CL, and number of follicular waves per interovulatory interval (IOI). For contralateral relationships, regardless of number of waves the diameter of the DF of wave 1 decreased (P < 0.03) between Days 11 and 13 when referenced to the follicle–CL relationship of wave 1 and decreased (P < 0.008) between Days 9 and 11 when referenced to the preovulatory follicle (PF)–CL relationship. For wave 2 in two-wave IOIs, the CL ovary of ipsilateral relationships had more (P < 0.05) follicles that reached at least 6 mm than the non-CL ovary. In three-wave IOIs, frequency of IOIs with the DF in the CL ovary was greater (P < 0.02) for wave 2 than for wave 3. In wave 3, the preovulatory and the largest subordinate follicles were located more frequently (P < 0.005) in the contralateral ovary. Ovulation in two-wave IOIs occurred more frequently (P < 0.0009) from the right ovary. In three-wave IOIs with a contralateral relationship ovulation occurred more frequently (P < 0.003) from the left ovary; a negative intraovarian effect of the CL on location of the PF may account for more ovulations from the left ovary and a reported greater frequency of the contralateral relationship. The hypothesis was supported that the ipsilateral versus contralateral relationship between the PF and CL is affected by the DF–CL relationship during the previous follicular waves and by the number and identity of waves per IOI.     

Estradiol-induced follicular atresia in rhesus monkeys is not prevented by exogenous gonadotropins

Estradiol-17β (E₂) induces atresia of the dominant preovulatory follicle (DF) when administered on day 6 of the menstrual cycle. The present study was designed to determine whether the atretogenic effect of E₂ could be averted by the administration of exogenous gonadotropins, in an attempt to determine whether E₂-induced atresia in primates is due to a direct action at the ovarian level or is mediated via pituitary secretion. After identification of the DF during laparoscopy, cyclic monkeys received Silastic capsules containing E₂ placed s.c. for 24 hours, plus one of the following treatments: phosphate-buffered saline, or 25 I.U. of either human urinary menopausal gonadotropin (hMG), FSH-rich hMG, human urinary FSH (uFSH), or human pituitary FSH (pFSH) injected i.m. twice daily for 2 days. The control treatment resulted in atresia of the DF and extended follicular phases (26.3 ± 5.9 days, x? ± S.D.), but in normal luteal phases following ovulation of a substitute DF. Similar results occurred in all animals receiving FSH-rich hMG or pFSH, and in 11 of 16 animals receiving hMG or uFSH (P > 0.05). Since all possible routes and regimens of gonadotropin administration were not attempted, a central action of E₂ cannot be ruled out. However, we believe that the experimental observations support our contention that the atretogenic action of E₂ is exerted in part at the ovary.