The Influence of Ovarian Stromal/Theca Cells During In Vitro Culture on Steroidogenesis,Proliferation and Apoptosis of Granulosa Cells Derived from the Goat Ovary

Early follicular development is closely related to oocyte‐granulosa cells‐ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis‐related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.     

The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals

Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.     

Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte‐cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1‐4) were determined. The obtained results confirmed the follicle‐type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1‐4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.     

Immunohistochemical localization of inhibin and steroidogenic enzymes in the ovary of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina)

To study the ovarian function of the Order Scandentia, the localization of inhibin and steroidogenic enzymes (3 beta-hydroxysteroid dehydrogenase/isomerase and aromatase) in the ovaries of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina) was immunohistochemically analysed. As in the results reported for other mammals, inhibin alpha-chain was localized in the follicular epithelium of secondary or Graafian follicles in the two species. The localization of aromatase in the ovary of these two species, however, was different. In the common tree shrew, the aromatase was localized in the thecal cells, whilst in other mammals it is localized in the granulosa cells. These results indicate that in the ovary of the common tree shrew, the oestradiol may be synthesized in the thecal cells.     

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.     

Secretion of inhibin in female Japanese quails (Coturnix japonica) from hatch to sexual maturity

To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.     

Comparison of aryl hydrocarbon receptor gene expression in laser dissected granulosa cell layers of immature rat ovaries

In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more β-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.     

Isolation of ovarian components essential for growth and development of mammalian oocytes in vitro

Mammalian ovaries contain a large number of oocytes, most of which degenerate either before or at various stages of growth. Dynamic and precise regulation in the ovary involves many factors, each with a unique role. Identifying the single most important factor is impossible; however, it may be possible to identify factors essential for oocyte growth. It is evident that oocytes can grow into competent ova in vitro; however, how faithfully the follicle should mimic the in vivo conditions remains unclear. In the culture system discussed in this review, bovine and mouse oocyte-granulosa cell complexes, at approximately the late mid-growth stage, spread on a substratum without the involvement of theca cells. The structural simplicity of this system is advantageous because it reduces the basic conditions essential for regulation of oocyte growth. Apart from biological factors, high concentrations of polyvinylpyrrolidone (molecular weight: 360000) improved oocyte growth. Among ovarian factors, androstenedione was used to compensate for the absence of theca cells, and it promoted both follicular growth and acquisition of oocyte meiotic competence. Most oocytes cultured in a group were viable after long-term culture, suggesting that unlike ovarian events, there was no exhaustive follicle selection. Collectively, oocytes and their associated granulosa cells can establish independent units capable of supporting oocyte growth in appropriately modified culture media.     

Ovine follicular fluid inhibits aromatase activity

“Within follicle” regulations may be important for the fine tuning of gonadotrophin action in ovarian follicles. While numerous growth factors, steroids or proteins which are present in follicular fluid have been shown to have the ability of positively or negatively affecting follicle function, the net effet of follicular fluid of the dominant follicle on its function is unclear.

A bioassay measuring aromatase activity of follicular walls was used (1) to check whether follicular fluid from dominant follicles can alter aromatase activity (2), to check how follicle size, atresia and specific gonadotrophins alter the effects of follicular fluid (3), to identify the nature (steroid or protein) of the active compound(s), and (4) to check whether the inhibition is specific of aromatase. Dominant follicular fluid had the ability to reduce aromatase activity. This effect was dose dependent and was obvious whether or not a protease inhibitor was added to the incubation medium. There was no difference in the magnitude of the inhibitory effect of follicular fluid when FSH (2 ng/ml) or no FSH was added to the incubation medium. LH, however, could potentialise the inhibitory effects of follicular fluid. Dominant follicular fluid was more potent to inhibit aromatase than follicular fluid from atretic follicles. Medium conditioned by granulosa cells, but not by theca cells could inhibit aromatase activity when added to the incubation medium. Charcoal treatment of dominant follicular fluid did not remove its inhibitory potential. Fractionation of dominant follicular fluid by a desalting column demonstrated that the inhibition was related to a compound(s) > 10 kDa. Finally, the effect of dominant follicular fluid on aromatase appears specific of this enzyme as follicular fluid does not affect androgen output by thecal shells or progesterone output by luteal cells.

Further research is required to check whether the activity observed in dominant follicular fluid is related to compounds known to affect aromatase activity (inhibin, mullerian inhibiting substance, heat shock protein 90, superoxyde dismutase) or to another peptide/protein.     

Intraovarian localization of growth factors in induced cystic ovaries in rats

We hypothesized that the special hormonal environment present in animals with cystic ovarian disease (COD) interferes with cellular production of growth factors (GFs). The objective of the present study was to characterize the expression of insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in induced COD using immunohistochemistry. We used an experimental model based on the exposure to constant light of adult rats during 15 weeks. We quantified the expression of GFs in cystic and normal ovaries by the Immunohistochemical Stained Area (IHCSA). In animals with COD, a significant reduction in the IHCSA of IGF-I in the follicular fluid, theca and granulosa layers of cysts occurred; and an increase in the interstitial tissue with regard to the control group. We found moderate immunoreactivity of FGF-2 in granulosa and theca layers of secondary and tertiary follicles and lower expression in the granulosa and theca interna layers of cystic follicles. Immunoexpression of VEGF was found in granulosa and theca cells of secondary and tertiary follicles. This study shows changes in the ovarian expression of IGF-I, FGF-2 and VEGF in induced COD. We can propose that an alteration in the control of the follicular dynamic, through the GFs, added to other features, could be involved in the ovarian cyst pathogenesis.     

促卵泡素对鸡等级前卵泡细胞发育的影响

实验研究了鸡等级前卵泡颗粒细胞和膜细胞的发育及促卵泡素(FSH)对其增殖的调控作用。形态学观察显示小白卵泡只有1层颗粒细胞,大白卵泡出现2层颗粒细胞,而小黄卵泡和大黄卵泡中则有多层颗粒细胞。结果表明:卵泡颗粒细胞和膜细胞的密度和细胞层厚度随着卵泡发育的等级而增加。卵泡体外悬浮培养表明,FSH显著刺激小黄卵泡和大黄卵泡中颗粒细胞的增殖,但对膜细胞无显著促增殖作用。由此推测,FSH通过刺激颗粒细胞的增殖促使等级前卵泡进入等级发育。     

生殖激素控制卵泡细胞凋亡的研究进展

研究表明颗粒细胞凋亡是导致卵泡闭锁的重要原因，而颗粒细胞凋亡涉及许多因素，其中生殖激素，如GnRH、FSH、LH、P4、E、A、GH、Mel、inhibin、activin、follistatin等间接地和直接地对卵巢卵泡细胞凋亡发挥重要的综合控制作用，因此正确理解激素对体内、外卵泡及颗粒细胞发育和衰亡的调节网络具有很重要的理论和实践意义。     

Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.     

Preliminary evaluation of influence of zearalenone on cocultures of granulosa and internal theca cells of ovarian follicles in bitches in in vitro culture

Zearalenone (ZEA) is an undesirable substance in feed materials and feed of plant origin. It is an example of the micotoxin that causes disturbances in the functioning of the reproductive system. The wide range of plant compounds in pet food means that ZEA may frequently have a negative effect on pet reproduction. An assessment of the influence of ZEA on the granulosa and theca cells of the ovarian follicle in bitches in vitro was carried out. The co-culture of the ovarian follicles was incubated for 72 hours with the addition of 12.5 ng/ml and 25.0 ng/ml of ZEA. Numerous vacuoles in the cytoplasm of the granulosa cells were noted in the culture with the addition of 25.0 ng/ml of ZEA. Preliminary investigations suggest negative effect of ZEA on the granulosa cells in bitches in vitro.     

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.     

Activins and Inhibins: Expression and Role in Normal and Pathological Canine Reproductive Organs: A review

Activins and inhibins are regulatory proteins of the reproductive function. Inhibins antagonise the activin signalling at different levels and are responsible for the negative feedback in the regulation of the release of pituitary follicle stimulating hormone (FSH), which, in turn, is promoted by locally produced activins. In the canine ovary, both peptides are expressed by developing follicles and corpora lutea. Activins may play a stimulatory role in follicular development, promoting the aromatase function; inhibins modulate these processes and suppress the hyperplasic/neoplastic stimuli. Activins are required for ovulation and corpus luteum formation, while inhibins stimulate progesterone synthesis. The exclusive production of alpha‐inhibin by granulosa cells allows the peptide to be used as marker to identify canine ovarian stromal tumours by immunohistochemistry. In the male, activins are powerful morphogenetic factors in the foetal testis. In the adult, they display a modulating action on spermatogenesis and Sertoli cell function. Inhibins, produced mainly by Leydig cells, promote testosterone secretion. Canine testicular tumours, such as Leydig, Sertoli and granulosa cell tumours (GCTs), may express inhibin subunits and produce high circulating levels of these glycoproteins. In the canine prostate, activins inhibit epithelium proliferation, antagonising androgen effects, but they are synthesised under androgenic stimulus.     

Expression of Osteopontin (OPN) mRNA in Bovine Ovarian Follicles and Corpora Lutea

The matricellular protein osteopontin (OPN) plays a role in various physiological processes, including angiogenesis and tissue remodelling. As these processes are essential for the maintenance of ovarian physiology, the aim of the study was to investigate the expression of OPN (mRNA) in ovarian cells and to evaluate whether it can be regulated by gonadotrophins. Using conventional RT‐PCR and real‐time PCR, we have detected and quantified OPN mRNA as well as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA expression in bovine granulosa, theca and luteal cells. In all cells examined, both genes were found in equal amounts and no striking variations in the expression could be observed between granulosa, theca and luteal cells. Furthermore, no effect on either OPN or GAPDH mRNA expression was evident after culturing ovarian cells in the presence of gonadotrophic hormones, although the cells were still highly responsive in terms of cAMP formation. Although neither variations between different cell types nor a regulation of OPN mRNA expression by gonadotrophic hormones could be detected, the high and unambiguous mRNA expression in steroidogenic cells suggests that OPN should be added to the growing list of intraovarian factors which may be involved in ovarian physiology.