Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

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     

Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4-6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17 beta (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH. 0.25 microgram/mL) or luteinizing hormone (LH. 1 microgram/mL). Addition of testosterone or androstenedione (0.5 microM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3'.5'-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.     

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.     

Determination of onset of apoptosis in granulosa cells of the preovulatory follicles in the bonnet monkey (Macaca radiata): correlation with mitogen-activated protein kinase activities

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48-72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

The effect of an LH pulse on 3H-thymidine incorporation into cultured ovaries of metestrous rats

Summary The effect of an LH pulse on the rate at which ³H-thymidine is incorporated into cultured ovaries of metestrous rats was studied. In comparison to ovaries cultured with tonic LH, an LH pulse (1) rescued follicles from atresia, (2) induced thecal cell proliferation, and (3) increased the rate at which granulosa cells enter mitosis. It is concluded that LH pulses increase follicular growth by first triggering thecal cell proliferation and then inducing mitotic divisions within the granulosa cells of both atretic and non-atretic follicles.     

Identification of cells migrating from the thecal layer of ovarian follicles

In the mammalian ovary, oocytes are contained within ovarian follicles. These consist in an oocyte surrounded by supporting cells: an inner layer of granulosa cells and an outer layer of thecal cells separated by a basal lamina. At any one time, a developing cohort of follicles exists, from which only a small species-specific number are selected for continued development towards ovulation, with the remainder dying by follicular atresia. Here, we use in vitro methods to study interactions between two follicles in culture (follicle co-cultures). We show that, when two individual follicles are grown together in culture, cells and cellular processes migrate from the outer thecal layer of one follicle to the thecal layer of the other co-cultured follicle. These cells are identified as a mixed population containing primarily endothelial but also neuronal cells. Both are able to migrate through the ovarian interstitum, making contact with the basal lamina of other follicles and with similar cells from these other follicles. Networks of such cells might be involved in interfollicular communication and in the coordination of follicle selection for ovulation.     

Gonadotropin regulation of rat ovarian lysosomes: Existence of a hormone specific dual control mechanism

Gonadotropic hormones PMSG (15 IU/rat), FSH (3 g/rat), LH (9 g/rat) and hCG (3 g/rat) were shown to decrease the free cytosolic lysosomal enzymes during the acute phase of hormone action in rat ovaries. When isolated cells from such rats were analyzed for the cathepsin-D activity, the granulosa cells of the ovary showed a reduction in the free as well as in the total lysosomal enzyme activities in response to FSH/PMSG; the stromal and thecal compartment of the ovary showed a reduction only in the free activity in response to hCG/PMSG. The results suggest the presence of two distinct, target cell specific, mechanisms by which the lysosmal activity of the ovary is regulated by gonadotropins.Abbreviations PMSG pregnant mare serum gonadotropin - FSH follicle stimulating hormone - LH luteinizing hormone - hCG human chorionic gonadotropin - GC granulosa cells - S/T stromal and thecal cells     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.