Hypertrophied Granulosa Cells Research Articles

Complementary histological and genomic analyses reveal marked differences in the developmental trajectories of ovaries in Siberian hamsters raised in long- and short-day lengths

Siberian hamsters (Phodopus sungorus) delay sexual development when raised in short-day (SD; 10 hr light: 14 hr dark) conditions, which leads to delayed onset of estrous cycles and ovulations as compared to females raised in long-day (LD; 16 hr light: 8 hr dark) conditions. In addition to the absence of pre-ovulatory follicles and corpora lutea, the ovaries of SD-reared Siberian hamsters are characterized by an abundance of hypertrophied granulosa cells (HGCs) that surround atretic oocytes. To determine the age at which the histology of LD and SD ovaries first diverge, including the initial appearance of HGCs in SD conditions, we examined hamster ovaries histologically at 1, 2, 3, 4, 6, 8, 10, and 12 weeks of age. After identifying subtle differences in LD and SD ovarian histology at 4 weeks of age, we searched for differences in ovarian gene expression at 3 and 8 weeks of age, which correspond to the ages when ovarian histology do not differ (3 weeks) versus the earliest age when HGCs were observed (8 weeks). At 3 weeks, only 14 genes were differentially expressed in LD and SD ovaries, whereas 183 genes were differentially expressed at 8 weeks. Overall, our findings demonstrate that ovarian development under SD conditions is not simply arrested at an early stage of LD development, but rather utilizes a developmental path that is distinct from that used in LD ovaries.

Photoperiod-gonadotropin mismatches induced by treatment with acyline or FSH in Siberian hamsters: impacts on ovarian structure and function

Many seasonal breeders time their reproductive efforts to specific times of the year to ensure adequate resources for the production and care of young. For long-day (LD) breeders, females born before the summer solstice (LDs) reach sexual maturity quickly and often breed that same year, whereas females born after the summer solstice (short days (SDs)) may delay reproductive development to the following spring when environmental conditions are favorable for reproduction. In Siberian hamsters, development in SD is associated with structural and functional differences in the ovary compared with females held in LD, including a greater number of primordial follicles and an abundance of hypertrophied granulosa cells (HGCs), which are immunoreactive for anti-Müllerian hormone. The goal of this study was to determine whether SD-induced gonadotropin suppression is responsible for these phenotypic differences. Gonadotropin levels were suppressed in LD hamsters using the GNRH antagonist acyline. Conversely, to determine whether the SD ovarian phenotype is completely reversed by gonadotropin stimulation, recombinant human FSH (rhFSH) was administered. Our treatments were successful in mimicking FSH concentrations of the opposite photoperiod, but they did not produce a comparable change in the ovarian phenotype. Most notable was the lack of HGCs in the ovaries of acyline-treated LD females. Similarly, HGCs were maintained in the ovaries of SD females treated with rhFSH. Our data suggest that gonadotropins alone do not account for the SD ovarian phenotype. Future studies will determine whether SD-induced changes in other factors underlie these phenotypic changes.

Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus

Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

Enhanced serum oestrogen levels and highly steroidogenic, luteinized atretic follicles in the ovaries of the Djungarian hamster (Phodopus sungorus) kept under a short photoperiod from birth.

In contrast to the elaborate information available on the effects of the photoperiod on the testes of hamsters, little is known about the influence on their ovaries. This study aimed to describe the ovarian follicular development and steroid hormone production in Djungarian hamsters kept from birth under a short daylight regime. Female Djungarian hamsters (Phodopus sungorus) were kept under two different light regimes: (i) 16 h light:8 h darkness (long daylight; LD) and (ii) 4 h light:20 h darkness (short daylight; SD). They were killed at 28, 56 and 80 days after birth; blood and ovaries were collected. Ovaries were either fixed in Bouin's solution or frozen. Fixed material was dehydrated, embedded in paraffin, serially sectioned at 5 microm and stained with haematoxylin and eosin, whereafter all healthy and atretic follicles were classified and counted. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) was histochemically demonstrated in 10 microm sections of frozen ovaries. Serum oestradiol-17beta and progesterone levels were determined by RIA. The numbers of healthy preantral and antral follicles were higher in LD than in SD hamsters. Antral follicles did not significantly differ in number during development in LD hamsters, but they were completely absent from 80-day-old SD animals. In LD animals the number of apoptotic preantral follicles dramatically increased with age. In SD animals the numbers of apoptotic antral follicles strongly decreased with age, whereas numerous non-apoptotic follicles with luteinized granulosa cells and a degenerated oocyte appeared, and in increasing numbers with age. During development, moderate 3beta-HSD activity was present in interstitial cells, theca cells of healthy follicles, and in both theca and granulosa cells of degenerating follicles. Strong enzyme activity was found in the hypertrophied granulosa cells of luteinized atretic follicles. Mean serum progesterone values varied from 2 to 6 nmol/l and were not different in LD and SD hamsters. Mean serum oestradiol levels varied from 132 to 542 and 325 to 2353 pmol/l in LD and SD hamsters respectively. The highest oestradiol levels were found in SD animals at day 28 of development. Folliculogenesis was dramatically disturbed in Djungarian hamsters raised under a short photoperiod. These animals developed high serum oestradiol levels and numerous luteinized atretic follicles with highly steroidogenic granulosa cells, which appear to be the source of the increased serum oestradiol levels.

Proliferative activity of follicles and serum steroid concentration in Scotophilus heathi (vespertilionid bat) during periods of delayed ovulation

Follicular kinetics were studied using immunocytochemical localization of proliferating cell nuclear antigen (PCNA) and changes in circulating steroid concentrations to investigate the cause of prolonged survival of Graafian follicles and delayed ovulation in the vespertilionid bat Scotophilus heathi. PCNA immunoreactivity was limited to the granulosa cells and varied with stage of follicular development and reproductive phase. Two waves of follicular growth appear to be initiated in October and February. The first wave resulted in the appearance of some late antral follicles in November. These antral follicles grew slowly and differed from typical mammalian Graafian follicles, as the cells of their cumulus oophoricus were large and hypertrophied. The second wave of follicular development resulted in fresh recruitment and selection of dominant follicles in February. The steroid-hormone profile showed an unusually high concentration of circulating androstenedione from October to December. The estradiol concentration showed two peaks coinciding with two peaks of PCNA immunoreactivity. The results show that the unusually high androstenedione concentration might be responsible for the development of non-ovulatory Graafian follicles with hypertrophied granulosa cells, which survive for a prolonged period. The decline in androstenedione concentration allows the development of ovulatory follicles in S. heathi.

Melatonin in the control of the estrous cycle of the Indian desert gerbil (Meriones hurrianae: Jerdon).

Daily administration of melatonin for 8 weeks, either in the morning (9 a.m.) or late in the afternoon (5 p.m.), resulted in significant prolongation of the estrous cycle in the Indian desert gerbil Meriones hurrianae. The gonadosomatic index increased significantly (p < 0.01) in the melatonin-treated gerbils compared to the control animals. Histological study of the ovaries revealed enlargement of follicles and corpora lutea with hypertrophied granulosa cells in the melatonin-treated gerbils in comparison to their controls. The data suggest that the mechanism involved in the gonadal response to melatonin may not be the same in the temperate and tropical species.

Effects of steroids and steroid antagonists on growth, gonadal development, and [formula omitted] ratios in juvenile steelhead trout

Steroids incroporated into Oregon Moist Pellets were fed to steelhead trout ( Salmo gairdneri) to determine if growth could be enhanced. In 1976, steelhead trout were treated with 17 α-methyltestosterone, diethylstilbesterol, diethylstilbesterol plus clomiphene citrate, 17 α-methyltestosterone plus flutamide (4′-nitro-3′ trifluoromethyliosbutranilide), or flutamide from the onset of exogenous feeding. In 1977, steelhead trout were started on treated diets 1 month after the onset of exogenous feeding and received testosterone propionate, testolactone, methylandrostenolone, 17 α-methyltestosterone, flutamide, estradiol, or estradiol plus progesterone. Fish treated with flutamide (20 μg/g food) or diethylstilbesterol (5 μg/g) plus clomiphene citrate (15 μg/g) from the onset of exogenous feeding showed an increase in weight compared with controls. The androgens and other estrogens tested did not enhance the growth of steelhead trout. All compounds (except flutamide) fed to the fish from the onset of feeding caused abnormal sex ratios. Androgens induced more males while estrogens induced more females. 17 α-methyltestosterone (1, 5, 15, or 35 μg/g), 17 α-methyltestosterone (5 μg/g) plus flutamide (20 μg/g), testolactone (10 μg/g), and methylandrostenolone (10 μg/g) advanced spermatogenesis. 17 α-methyltestosterone (2 or 35 μg/g), testolactone (10 μg/g), methylandrostenolone (10 μg/g), or flutamide (20 μg/g) caused hypertrophied granulosa cells or atretic oocytes in some ovaries. Intersexes containing both testicular and ovarian tissue in the gonads were found in some fish treated with 17 α-methyltestosterone (5 μg/g), 17 α-methyltestosterone (5 μg/g) plus diethylstilbesterol (5 μg/g), 17 α-methyltestosterone (5 μg/g) plus flutamide (20 μg/g), or diethylstiblesterol (5 μg/g) plus clomiphene citrate (5 μg/g). 17 α-methyltestosterone at 1, 5, 15, or 35 μg/g caused significant linear dose-dependent increases in epidermal thickness. There were no differences in the RNA DNA ratio between treatments. We conclude from these studies that steelhead trout do not respond positively in terms of growth enhancement from steroid treatment as has been shown in other salmonids. However, the androgen antagonist, flutamide, may be a potential growth enhancer in steelhead trout and may also be used as an antiandrogenic compound when administered with an androgen.

Steroid-synthesizing cells in the embryonic and adult gonads of the domestic pigeon, Columba livia (Gmelin)

Steroidogenic cells in the developing gonads of Columba livia from Day 9 of incubation till the bird attains maturity have been histochemically identified by demonstrating the presence of Δ 5-3 β-hydroxysteroid dehydrogenase (Δ 5-3 β-HSDH). In the embryonic ovary the medulla, containing Δ 5-3 β-HSDH-positive cells, gradually seems to migrate into the cortical region with growth of the embryo and later it dwindles in 15- to 20-day-old hatchlings. In later stages, Δ 5-3 β-HSDH is found in some cells of theca interna of normal follicles, hypertrophied granulosa cells of atretic follicles, the granulosa and theca interna of post-ovulatory follicles, and the interstitial stromal cells. On Day 9 of incubation in the male embryo the convoluted solid seminiferous cords show a weak Δ 5-3 β-HSDH activity. One day prior to hatching, Δ 5-3 β-HSDH activity disappears from the seminiferous cords but appears in the interstitial Leydig cells. In the 4-month-old adult pigeon, Δ 5-3 β-HSDH activity is seen in the Leydig cells as well as in the seminiferous epithelium.

Steroid synthesizing cellular sites in the ovary of catfish, Mystus cavasius: A histochemical study

The distribution of Δ 5-3 β-hydroxysteroid dehydrogenase (Δ 5-3 β-HSDH), 17β-hydroxysteroid dehydrogenase (17β-HSDH), 11β-hydroxysteroid dehydrogenase (11 β-HSDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities has been studied by histochemistry in the ovary of the catfish, Mystus cavasius. The enzyme activities occurred in the thecal cells of the ovary. In addition, a weak enzyme activity was observed in the hypertrophied granulosa cells of early atretic follicles. In later stages these phagocytic granulosa cells degenerated and only thecal cells remained in the connective tissue, surrounded by ovarian stroma. These thecal cells transformed into interstitial gland cells and an intense Δ 5-3 β-HSDH, 17β-HSDH, 11β-HSDH and G-6-PDH enzyme activity occurred in them. It is suggested that the thecal cells of mature follicles and the interstitial gland cells form important sites of steroidogenesis in the ovary of M. cavasius.

Histochemistry and ultrastructure of pre- and post-ovulatory follicles in the ovary of the goldfish, Carassius auratus

Pre- and post-ovulatory follicles in the goldfish ovary were investigated histochemically and ultrastructurally. Special cells in the thecal layer of preovulatory follicles of yolk-laden oocytes showed 3β-hydroxy-Δ5-steroid dehydrogenase (3β-HSD; EC 1.1.1.145) activity typical of steroid hormone synthesis. Ultrastructurally, these thecal cells possess organelles characteristic of steroid-producing cells: mitochondria with tubular cristae and agranular endoplasmic reticulum. Unlike thecal ceils, the granulosa cells contain organelles typical of protein-secreting cells. These findings strongly suggest that only the special theca cells are sites of estrogen synthesis in the goldfish ovary.Postovulatory follicles, 6–10 h after ovulation, were characterized by a highly vascular thecal layer and hypertrophied granulosa cells. A weak but definite 3β-HSD activity occurs in both special theca cells and granulosa cells. Ultrastructurally, the granulosa cells contain many lipid droplets and numerous Golgi elements, suggesting the possible immediate transformation of granulosa cells to lutein cells during the ovulation. However, 30 h after ovulation, both granulosa cells and special theca cells showed advanced degenerative features and these data do not seem to support the hypothesis of an endocrine role for the older postovulatory follicles of the goldfish.

Steroid-synthesizing cellular sites in amphibian ovary. A histochemical study

Ovaries from four oviparous Anura ( Rana cyanophlyctis, Rana tigrina, Rana verrucosa, and Cacopus systoma) were histochemically studied for the distribution of NADH and NADPH diaphorase, glucose-6-phosphate dehydrogenase (G-6-PDH) and Δ 5-3 β-hydroxysteroid dehydrogenase (Δ 5-3 β-HSDH). Both the diaphorases and the G-6-PDH were distributed throughout the ovary. A weak Δ 5-3 β-HSDH activity was demonstrated in the follicular cells of large previtellogenic, vitellogenic, and mature follicles and also in the interstitial cells. Δ 5-3 β-HSDH activity found in the granulosa lutein cells of the postovulatory corpora lutea was maximum on the day of spawning and decreased markedly thereafter and its disappearance was associated with the degeneration of these cells. During the early stage of atresia of vitellogenic follicles a very weak Δ 5-3 β-HSDH activity was found in the hypertrophied granulosa cells, but this soon disappeared.