Ultrastructural changes in granulosa lutein cells and progesterone levels during preimplantation,implantation, and early placentation in the western spotted skunk

Summary The ultrastructure of corpora lutea obtained during the preimplantation, implantation and early postimplantation periods has been studied in 20 western spotted skunks. Fine structure of granulosa lutein cells was correlated with progesterone levels. The corpus luteum of the prolonged (7 month) preimplantation period contained undifferentiated small granulosa cells and differentiated large granulosa lutein cells. The former ranged in size between 12 and 20 and the latter between 20 and 45 . The ratio of small and large cells was about equal in an animal 2 days prior to nidation whereas only few small cells and numerous large cells were observed in an animal estimated to be 8 to 12 hours from nidation. Occasionally small cells were observed amidst large ones during the 24 hour nidation period, i.e. adhesion of trophoblast with the luminal uterine epithelium, but small cells were absent in animals after this period. Small cells had some smooth and rough endoplasmic reticulum, rod-shaped mitochondria with platelike cristae, small Golgi complex, and relatively smooth plasma membranes. Large lutein cells had abundant smooth endoplasmic reticulum, membranous whorls of smooth endoplasmic reticulum, usually round mitochondria with tubular and lamellar cristae, a well developed Golgi complex, variable amounts of lipid droplets, and highly plicated and ruffled plasma membranes. Peripheral plasma progesterone levels during the prolonged preimplantation period ranged between 1.1 and 7.9 ng/ml, but during implantation it was between 8 and 16.6 ng/ml. It is suggested that plasma progesterone levels fluctuate during the time of implantation and should not be regarded as a basis to predict actual nidation in the western spotted skunk.This research was supported in part by Grant Number HD06556 from the National Institute of Child Health and Human Development.     

Partial development of the steroidogenic ultrastructural features in degenerative corpora lutea after a single injection of pituitary extract in the Western painted turtle (Chrysemys picta)

Pituitary glands were removed from sexually mature female turtles (Chrysemys picta) and they were injected intraperitoneally (i.p.) into other mature females of the same species (experimental). In addition mature females of the same species received saline injection only (controls). Initially all the turtles used in this study were steroidogenically inactive with corpora lutea already undergoing luteolysis (degeneration) as these turtles had ovioposited their eggs approximately 2 weeks earlier. Forty-eight hour post injection the corpora lutea were removed from the control and experimental turtles. In the experimental turtles, the lutein granulosa cells developed ultrastructural features such as tubular and cisternal smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae associated with lipid droplets. However, the controls maintained degenerative corpora lutea without steroidogenic ultrastructural features. The circulating progesterone (Pro) levels in the experimental turtles were significantly higher than the controls (P<0.049). Although the 48h development of steroidogenic ultrastructural features in the lutein granulosa cells was only partial in development, the effect of the pituitary taken from the inactive donor triggered an activating process within a short period, clear evidence of gonadotropic effect on the inactive corpora lutea. The present data offer interesting information on the short-term effect of gonadotropins during the non-reproductive period. This information may have useful implication under natural conditions particularly during the onset of a new reproductive cycle where the ovary is still inactive.     

The fine structure of the granulosa cells in the domestic fowl and the rat

Summary The granulosa cells of the ovarian follicle of the rat and the domestic fowl have been studied with the light and electron microscope. The nuclei of the granulosa cells were irregular with indentations and large in proportion to the cytoplasm of the cell. The mitochondria had a dense, dark matrix with only few cristac. The Golgi apparatus was moderately developed, located towards the oocyte in a juxtanuclear position. The endoplasmic reticulum was rather sparse. Lipid droplets were only occasionally encountered. Microtubules were regularly observed. The functions of the granulosa cells are discussed. Compared with the steroid-producing cells of the theca interna of the same follicles, the granulosa cells primarily are the nursing cells for the growing oocyte and mainly have the characteristics of protein forming cells.     

The effects of clomiphene on the granulosa cells of the domestic fowl

Summary The present paper describes for the first time the fine structure of ovarian granulosa cells as seen after administration of clomiphene to the domestic fowl for a 28 days period.The main cytoplasmic changes of the granulosa cells seemed to be an increase in the number of mitochondria, dense bodies and complex bodies. The Golgi apparatus became enlarged, and there was an increase in the endoplasmic reticulum, annular desmosomes and cytoplasmic processes.All the observations made are similar to those made after administration of steroids and gonadotropins. In conclusion, therefore, the present study has demonstrated that administration of clomiphene exerts a stimulating effect on the granulosa cells. The mechanism of this effect is discussed.     

The follicular oocyte and its granulosa cells in domestic pig

Summary Pig oocytes and their surrounding granulosa cells obtained from mature Graafian follicles at a stipulated time near to ovulation were studied in some details electronmicroscopically. Particular emphasis is given to the corona radiata cell processes and to the heterogeneous population of mitochondria in the oocyte.The corona radiata cell processes contain various components such as filaments, mitochondria, multivesicular bodies and lipid droplets in their matrix. The contact relationship of the corona radiata cell processes to the oocytes is maintained by desmosomes. Usually, the two parallel surface membranes forming the desmosome are separated by a space of about 200 Å. Occasionally, the two membranes approximate each other to form a junction having a gap of about 70 Å. Apparently the membranes become fused in some regions.Of particular interest is the distribution and structural characteristics of the single-membrane-bounded structures, and their relationship to the cytomembranes and the mitochondria. On the basis of the present and earlier (Norberg, 1972) observations, the question arises whether the formation and development of mitochondria of pig oocytes depend, at least partly, on a metamorphosis of single-membrane-bounded structures derived from less complex membraneous elements. Final conclusions concerning this problem demand integrated morphological and biochemical investigation regarding the biosynthesis of mitochondria.This work was supported by the Agricultural Research Council of Norway.     

Contribution à l'étude infrastructurale du système neurosécréteur rétrocérébral chez Locusta migratoria migratorioides (R. et F.)

Résumé L'étude infrastructurale des corpora cardiaca de Locusta révèle l'existence de 3 types de fibres neurosécrétrices (portion neurohémale) et d'un seul type de cellules glandulaires (portion endocrine intrinsèque). Elle permet également d'envisager les modalités du rejet des sécrétions exogènes et endogènes.

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Ultrastructural study of the retrocerebral neurosecretory system in Locusta migratoria migratorioides (R. et F.)I. The corpora cardiaca

Summary An ultrastructural study of the corpora cardiaca of Locusta migratoria migratorioides shows three neurosecretory fiber types in the neurohaemal part of these organs; only one cellular type constitutes the intrinsic glandular part of the corpora cardiaca. This study also shows sites of release of stored extrinsic neurosecretory material and of products elaborated by intrinsic glandular cells of the corpora cardiaca.

     

The pattern of ovulation in the southern African spiny mouse (Acomys spinosissimus)

The pattern of ovulation in mammals is generally considered to be either spontaneous or induced by coitus. The present study aimed to assess the pattern of ovulation in the southern African spiny mice (Acomys spinosissimus). Females were divided into three treatments differing in the degree of contact with a male. Control females had no contact with males, separated females had only chemical, auditory and visual contact with a male as the sexes were separated with wire mesh and paired females had full contact with a vasectomized male and copulations were possible. Each treatment consisted of seven females and the ovarian mass, the number of primary, secondary/tertiary and Graafian follicles as well as presence of corpora lutea were compared between the three treatments. Faecal progestagen metabolite (FPM) concentrations were analyzed for every second day throughout the experiment and they were used to determine luteal phases and oestrous cycles. Corpora lutea were found in both the control and the paired treatment indicating that ovulation also occurred in the absence of coitus. There was also no effect of treatment on ovarian mass or follicle numbers. In contrast, only females in the separated and paired treatments exhibited luteal phases and oestrous cycles. Especially at the beginning of the experiment, FPM concentrations were higher in those two groups than the control. The results indicate that A. spinosissimus appears to ovulate spontaneously, although physical as well as olfactory male cues appear to be of great importance to enhance reproductive efforts of females.     

The effects of steroids on the granulosa cells in the domestic fowl

Summary The fine structure of granulosa cells of the domestic fowl as seen after administration of steroids is described. Diaethylstilboestrol, estradiol and hydroxy-progesterone were given as intramuscular injections for a 28-days period. The main cytoplasmic changes of the granulosa cells were an increase in the number of mitochondria and dense bodies. The Golgi apparatus became enlarged, and occupied a large portion of the cell. The nucleus was found located adjacent to the oocyte, and there was an increase in number of the annular desmosomes. The investigation has demonstrated that even if steroids in high dosages induce atrophic alterations of the ovary, they stimulated proliferation of the granulosa cells of the small follicles. Apparently the small follicles with the granulosa cells retain the ability to regain development, which may be to some importance when steroids are used therapeutically (gynecologic disorders, contra-ception).     

Progesterone exposure of the preovulatory follicle in the seasonally anestrous ewe alters the expression of angiogenic growth factors in the early corpus luteum

Gonadotrophin releasing hormone (GnRH)-induced ovulation in seasonally anestrous ewes is associated with a high incidence of defective corpora lutea (CL), which can be completely eliminated by priming ewes with progesterone before GnRH treatment, but the physiological basis of this has remained elusive. This study tested the hypothesis that progesterone priming eliminates defective luteal function by altering the expression of Vascular Endothelial Growth Factor (VEGF), its receptor VEGFR-2, and angiopoietin (ANG)-1, ANG-2 and their receptorTIE-2 in the early CL. Fifteen seasonally anestrous ewes were treated by i.m. injection with 20 mg of progesterone 3 days before the start of GnRH treatment, while another 15 animals served as controls. Intravenous injections of 500 ng GnRH were given to all the ewes every 2 h for 28 h, followed by a 300 μg GnRH bolus injection to synchronize the preovulatory luteinizing hormone (LH) surge. Corpora lutea were collected 1, 2 and 4 days after ovulation and analyzed for protein and mRNA expression of VEGF, VEGFR-2, ANG-1, ANG-2 and Tie-2 using Western Immunoblotting and in situ hybridization. VEGF, VEGFR-2 and ANG-1 expression was significantly higher (P ≤ 0.05) in the CL of progesterone-primed animals compared to non-primed ones. However, no differences were observed in the ANG-2 or Tie-2 expression levels between the two treatment groups. These data suggest that progesterone priming of the preovulatory follicle alters the expression of some angiogenic growth factors in the early CL, leading to greater vascular stability and thereby normal luteal function.     

Short-lived corpora lutea syndrome in anoestrous ewes following 17β-oestradiol or MAP treatments applied before an allogenic sexual stimulation with rams and oestrous ewes

When induced to ovulate during anoestrus, ewes, does and cows frequently develop a short-lived corpora lutea (SLCL) syndrome associated to lack of previous progesterone. Exogenous progesterone precludes SLCL by blocking oxytocin endometrial receptors, thus inducing normal life-span CL (NLCL). Paradoxically, circa 50% of unprimed ewes do not develop SLCL. We report results from 3 trials assessing follicular, oestrous, ovulatory, and luteal end-points after 17β-oestradiol or MAP treatments. Oestradiol benzoate (50 μg) induced follicular turnover, provoked ovulation in 40% (24/60) of ewes treated (93% of which developed SLCL), but did not affect the incidence of SLCL (26/53) after an allogenic sexual stimulation (ASS) by rams and oestrous ewes. By the onset of the ASS, most NLCL ewes (26/27) had already experienced turnover of their largest follicle, had smaller largest and second largest follicles, and ovulated their largest follicle more frequently than SLCL ewes did. Most SLCL ewes (19/25) did not ovulate their largest follicles, ovulating instead smaller follicles of identical size to those of NLCL ewes. Priming (40 mg of MAP for 12 days) was partially effective at preventing SLCL even when terminated 14 days in advance of an ASS, but failed at completely preventing SLCL when terminated 6 or more days in advance. The coupling of a timed acquisition of full steroidogenic capability before ovulation with a system of endometrial oestradiol–progesterone–oxytocin receptors linked in an unstable equilibrium controlling the amplification of the luteolytic feed-forward loop of oxytocin and prostaglandin F₂α explains occurrence and relative incidences of both NLCL and SLCL, and links proximate and ultimate causes of the SLCL syndrome.     

Ultrastructure of corpora allata of known activity during the vitellogenic cycle in the cockroach Diploptera punctata

Summary Ultrastructure was correlated with rates of juvenile hormone synthesis in corpora allata from females of the viviparous cockroach Diploptera punctata at seven daily intervals during the first vitellogenic cycle. Synthetic activity of the glands was determined by in vitro radiochemical assay before the glands were fixed for electron microscopic analysis. The cycle in rates of juvenile hormone synthesis progressed from about 20 pmol h^-1 per gland pair (oocytes 0.60 mm long) to a maximum mean rate of 140 pmol h^-1 per pair (oocytes 1.40–1.47 mm long) and declined to about 20 pmol h^-1 per pair at ovulation (oocytes about 1.65 mm long). Conspicuous ultrastructural changes occurred with changing synthetic rates. In glands with increasing rates of synthesis, mitochondria showed less electron-dense matrix, greater diameter and more irregular shape. Smooth endoplasmic reticulum changed from easily seen to obscure tubules, networks, and vesicles. Rough endoplasmic reticulum appeared in longer, more curved segments. Newly formed autophagic vacuoles appeared in all glands of highest activity rates. In glands with decreasing rates of synthesis, the mitochondrial matrix became denser, width smaller, and shapes less irregular. Smooth endoplasmic reticulum again appeared tubular and distinct. Golgi complexes were more conspicuous. Rough endoplasmic reticulum in whorls and large numbers of autophagic vacuoles continued to be present.This work was supported by USPH Grant AI 15230. We thank Kuen-Kuen Chan for skillful and thoughtful technical assistance     

Hamster oocyte penetrability during preovulatory maturation

In vitro fertilization techniques were used to analyze the penetrability of preovulatory hamster oocytes. The zonas of granulosa cell-free primary (GV) oocytes were penetrated in vitro in 2-3 h as readily as those of ovulated secondary oocytes (80% vs. 88%), whether inseminated separately or as mixed oocyte groups. In fact, a significantly higher (P less than 0.05) mean number of perivitelline spermatozoa was present in immature (3.6) compared with secondary (1.9) oocytes, primarily reflecting a lack of the zona block to polyspermy in the immature population. By contrast, when granulosa cells remained around GV oocytes, zona penetration was low and more were penetrated, with more spermatozoa incorporated into the vitellus as a function of increasing time of oocyte recovery after hCG. We conclude, contrary to previous reports, that the zona pellucida of the hamster GV oocyte is readily penetrable by spermatozoa in vitro. However, the resumption of meiosis brings an increase in the penetrability of the granulosa cell vestment as well as the capacity for cortical granule exocytosis and the ability to decondense and transform the fertilizing sperm nucleus. The fact that the zona pellucida of the immature oocyte has proved to be penetrable in vitro and/or in vivo in all the mammals studied in this respect is discussed with particular reference to the situation in man.     

中国大鲵闭锁卵泡的显微和超微结构观察

用光镜和电镜观察了中国大鲵卵泡闭锁过程和闭锁小体的显微和超微结构。结果显示 ,大鲵闭锁小体是卵泡细胞侵噬卵母细胞并增殖形成细胞团 ,膜细胞未参与。在大部分卵泡处于缓慢生长期时 ,未发现卵泡闭锁现象 ;在 5、 6月份 ,卵巢内大部分卵母细胞进入卵黄形成前期 ,部分卵泡闭锁 ,但闭锁小体细胞的类固醇激素分泌结构特征不明显 ;在 7、 8月份 ,大多数卵母细胞处于卵黄形成期 ,闭锁小体细胞具有管泡状嵴线粒体、丰富的滑面内质网和脂滴、发达的高尔基体等。这些细胞学特征表明闭锁小体可分泌类固醇激素 ,以调节正常卵子的成熟。在大鲵中观察到的闭锁小体属于排卵前黄体     

PTEN expression in ovine granulosa cells increases during terminal follicular growth

In the present paper, we have studied the expression of the Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and its putative biological role in the sheep ovary. We found by Northern-blot, immunohistochemistry and immunoblot that PTEN is highly expressed in granulosa cells from large differentiated follicles (LF) in comparison with small proliferating follicles (SF) (P < 0.001), with no clear effect of follicle quality. Moreover, the PTEN lipid phosphatase activity is also higher in LF than in SF (P < 0.01). In contrast, levels of the phosphorylated form of AKT (pAKT) are lower in LF than in SF (P < 0.0001). IGF-I and insulin but not FSH, LH or forskolin are able to stimulate the expression of PTEN mRNA (P < 0.001) and protein by ovine granulosa cells after 48 h of culture in vitro. An IGF-1 time course analysis showed that expression of PTEN protein appeared after 12h of culture, concomitant with the fall of the pAKT levels, which peaked after 6h of stimulation with IGF-I. Moreover, transfection experiments showed that overexpression of PTEN in ovine granulosa cells induced a decrease and an increase in E2F and p27 promoter activity, respectively (P < 0.05). Overall, our present data show for the first time that the expression of PTEN increases during terminal follicular growth. This increase, that might be induced by IGF-I but not FSH, would participate in the proliferation/differentiation transition of ovine granulosa cells in differentiating follicles.