Granulosa Cells Of Secondary Follicles Research Articles

P-649 Unconventional action of Luteinizing Hormone

Abstract Study question Has Luteinizing Hormone (LH) a role in the activation/survival of follicles of the primary ovarian reserve? Summary answer LH promotes survival and perhaps activation/growth of primordial and primary follicles, proliferation of granulosa cells of secondary follicles, reduces follicle atresia at later growing stages. What is known already In the mammalian ovary, Primordial Follicles (PMFs) constitutes the primary ovarian reserve, which declines over time due to physiological events such as atresia, recruitment, and ovulation. While in the adult, the role of LH in steroidogenesis and ovulation is well established, recent work from our laboratory has shown that under chemotherapeutic treatments, LH supports PMFs survival in prepuberal mouse ovary. Study design, size, duration LH effects on follicle dynamics and some activities of pre-granulosa cells were investigated in vitro and in vivo. In vitroexperiments were performed on PD4 GFP/Kit mice ovarian fragments and on PD8 CD1 mice ovarian somatic cells cultured up to six days in continuous presence of 0.2, 0.5, 1IU LH. In vivo experiments were performed on 9 PD4 and 8 PD28 CD1 mice receiving an intra-peritoneal injection for eight consecutive days of 1-5IU LH. Participants/materials, setting, methods Number and diameters of the GFP stained oocyte were counted in the ovarian fragments. Kit-Ligand and LH-R mRNA expression levels were analyzed by qPCR in pre-granulosa cells. For in vivo experiments, ovarian volume was measured by Micro-Ct and follicle counts were performed on histological sections. Estrous cycle phases were monitored by vaginal smear during the 8 days of treatment. Statistics was performed using Student’s t-test. Main results and the role of chance In fragments of PD4 mouse ovaries cultured in vitro for six days, 1IU LH significantly increased the number of oocytes with a diameter >40 mm (CTRL 17.7% vs LH 36.07%, p < 0,05), suggesting activation of PMFs. Moreover, the hormone increased the expression of mRNA for LHR and KL in the somatic ovarian cells and promoted their proliferation in vitro. The effects of 1IU LH on PD4 females can be summarized as follows: 1. Ovary volume increase (CTRL 0.3 mm3 vs LH 0.36 mm3, p = 0.07), 2. higher percent of PMFs (CTRL 68.227% vs LH 78.29%, p < 0,05) associated with lower percent of primary (CTRL 7.2% vs LH 4.05%, p < 0.04) and growing follicles (CTRL 14.18% vs LH 8.56%, p = 0.1), 3. higher number of granulosa cell layers (> three layers) in secondary follicles (CTRL 42.8% vs LH 54.6%), and 4. lower percent of atretic follicles (CTRL 2.64% vs LH 1.72%, p = 0.1). Similar results were obtained on 28 dpp mice. Morever, we observed that corpora lutea were present only in ovaries of LH-treated females and that all such females were in estrus. Limitations, reasons for caution The study was conducted only in mice. Wider implications of the findings These studies have highlighted new possible roles of LH on follicle classes (primordial/primary) whose development is considered gonadotropin independent. The results, if confirmed in humans, imply that LH might be used as a treatment for patients with ovarian disorders involved the survival/dynamics of the ovarian reserve. Trial registration number NA

Metabolic milieu and localization of ovarian leptin and receptor in queens under different reproduction phases.

Metabolic profile and leptin presence and expression of receptor in the ovary of 8 queens were analyzed. All queens were assessed for nutritional state and underwent ovariectomy in accordance with Italian law for the control of stray cats and dogs. Three queens were at early gestation, three prepubescent and two non-pregnant. All animals appeared healthy but underweight. Haemogram and biochemical profile did not show to be of prognostic value. On microscopy, granulosa cells of secondary follicles tested positive to leptin in pregnant queens, while testing negative in non-pregnant ones. Muscular layer of vessels of the ovary in non-pregnant queens tested positive to receptor, but negative in those found pregnant. Results suggest that leptin is produced and receptor expressed in the different tissues of the ovary in underweight queens. Additionally, the expression of receptor did not necessarily imply local leptin production and clearly displayed to vary according to the reproduction phase. Taken altogether, these findings allow to conclude that the expression of receptor can be interpreted as the pillar bridging ovary tissues as target sites of circulating leptin produced elsewhere. Arguably, ovary removal may affect hormonal cross-talk at the systemic level, likely affecting different systemic functions, including the nutritional status.

Receptors for thyroid-stimulating hormone and thyroid hormones in human ovarian tissue

Dysfunction in thyroid regulation can cause menstrual and ovulatory disturbances, the mechanism of which is not clear. The distribution and activity of the thyroid-stimulating hormone (TSHR), and the thyroid hormone receptors (TR) alpha1, alpha2 and beta1 in human ovarian tissue and in granulosa cells was studied using immunohistochemistry, reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative PCR and immunoassays. Strong immunostaining of TSHR, TRalpha1 and TRbeta1 was observed in ovarian surface epithelium and in oocytes of primordial, primary and secondary follicles, with minimal staining in granulosa cells of secondary follicles. Granulosa cells of antral follicles expressed TSHR, TRalpha1 and TRbeta1 proteins. Messenger RNA for all receptors was present in ovarian tissue. Mature human granulosa cells expressed transcripts for 5' deiodinases types 2 and 3, but not type 1, indicating the possibility of conversion of peripheral thyroid hormone thyroxin (T(4)). Granulosa cells stimulated with TSH showed a significant increase in cAMP concentrations after 2 h of culture (P = 0.047), indicating activation through TSHR. Stimulation with T(4) resulted in increased extracellular signal-regulated kinase 1 and 2 activation after 10, 30, 60 min and 24 h. These data demonstrate that TSH and thyroid hormone receptors may participate in the regulation of ovarian function.

Cell‐specific Localization of Oestrogen Receptor beta (ESR2) mRNA within Various Bovine Ovarian Cell Types Using In situ Hybridization

The localization of oestrogen receptor beta (ESR2) mRNA, in this article denominated as (ERbeta) mRNA, was examined using in situ hybridization in the ovaries of randomly selected cows, irrespective of the cycle stage of the animals. A 602-bp fragment of ERbeta mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Semi-quantitative evaluation showed that the scores for ERbeta mRNA were moderate to high in the follicle cells of both primordial and primary follicles, but lower in granulosa cells of secondary follicles. In vital tertiary follicles, the total ERbeta mRNA expression was low but varied between the different animals. In both obliterative and cystic atretic follicles, high to moderate ERbeta mRNA scores were noticed in the granulosa cells. The stroma cells surrounding primordial and primary follicles and the theca cells of secondary follicles showed moderate ERbeta mRNA levels, whereas the ERbeta mRNA score in theca interna and theca externa cells of vital tertiary follicles was distinctly higher. In the theca cells of atretic follicles the score was even higher. Cells of corpora hemorrhagica and corpora lutea had moderate ERbeta mRNA scores, while higher scores were seen in cells of corpora albicantia. Cells of the surface epithelium had a moderate score for ERbeta mRNA, whereas cells of the tunica albuginea and deep stroma showed high ERbeta mRNA scores. The present findings have clearly established a cell-specific localization of ERbeta mRNA in several cell types in the bovine ovary.

Changes in Expression and Localization of Connexin 43 mRNA and Protein in Porcine Ovary Granulosa Cells during Follicular Atresia

Gap junctions contain channels that connect neighboring cells by allowing the movement of molecules smaller than 1,200 Da. They are formed by connexins and may play a crucial role in the regulation of apoptotic cell death. To determine the role of connexin 43 (Cx43), which is dominantly expressed in granulosa cells, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression and localization of Cx43 mRNA and protein in granulosa cells during atresia using the quantitative real-time revese transcription-polymerase chain reaction, in situ hybridization, Western blot, and immunohistochemistry. Stages of follicular atresia were assessed based on histochemical terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) and/or the ratio of progesterone and 17beta-estradiol levels in follicular fluid measured by radioimmunoassay. Cx43 mRNA was detected in granulosa cells of secondary follicles and of healthy, early and progressed atretic tertiary follicles, but not in those of primordial or primary follicles. Both phosphorylated/activated and non-phosphorylated/native Cx43 proteins were detected in granulosa cells of secondary and tertiary follicles, but not in those of primordial or primary follicles. Moreover, in tertiary follicles, these Cx43 proteins were expressed most strongly in granulosa cells of healthy follicles, but only trace levels were noted in cells of early atretic and progressed atretic follicles, an indication that the expression levels of Cx43 protein decrease during follicular atresia. These findings indicate that Cx43 is involved in the apoptosis of granulosa cells during atresia in porcine ovaries.

SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis.

In the present study, we screened for subtilisin-like proprotein convertases (SPCs) that potentially regulate the activation of known growth factors during embryonic development. We isolated a novel protease, SPC7, as well as several known SPCs. SPC7, like SPC1, is expressed ubiquitously throughout development. In contrast, SPC4 and SPC6 exhibit dynamic expression patterns. SPC4 transcripts were initially detected in the granulosa cells of secondary follicles. Shortly after implantation, SPC4 transcripts are localized to extraembryonic cell populations, and at later stages are detected in discrete tissues including the primitive gut, heart, neural tube, and limb buds. Within the limb buds, SPC4 mRNA is most abundant in the apical ectodermal ridge (AER). At later stages of limb development, SPC4 mRNA is strongly expressed in cartilage and in the interdigital mesenchyme. In contrast, high SPC6 mRNA levels are detected in somites, the dorsal surface ectoderm, and in vertebral cartilage primordia. In limb buds, SPC6 is strongly expressed in the AER, and at later stages in dorsal mesenchyme. A comparison of these expression patterns with those of several bone morphogenetic proteins (BMPs) indicates that processing of these growth factors may be limited by the local availability of SPCs.

Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.

Cyclic Changes in Follistatin Messenger Ribonucleic Acid and its Protein in the Rat Ovary During the Estrous Cycle*

The purpose of this research was to characterize the localization of follistatin mRNA and protein in the adult rat ovary during the 4-day estrous cycle. Analysis of ovarian sections using in situ hybridization and immunohistochemistry demonstrated the presence of follistatin messenger RNA (mRNA) and its protein in granulosa and luteal cells; no follistatin (message or protein) was detected in any of the other ovarian cell types. An important observation was that the intensity of follistatin signals changed during granulosa differentiation and the estrous cycle. During folliculogenesis, the first detectable hybridization signal appeared in the granulosa cells of secondary follicles, but the signal was weak. However, when a preantral follicle reached the early tertiary stage (beginning antrum formation), the message signal was very strong, being expressed in all granulosa cells of all such follicles (300-400 microns in diameter). In atretic follicles, follistatin mRNA was localized to granulosa cells, but only during the early stages. The above hybridization pattern of follistatin mRNA in prenatral and atretic follicles appeared constant throughout the estrous cycle. Interestingly, immunohistochemistry studies showed that the follistatin protein was detected only in certain follicles, being restricted to those which were healthy. On the morning of estrus, the follistatin protein was localized to a subpopulation of early tertiary follicles, presumably the dominant follicles selected to ovulate in the next cycle. As the dominant preovulatory follicles matured through diestrus and proestrus, the follistatin mRNA and protein signals appeared more intense in the granulosa cells. After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpora lutea on estrus morning. After luteolysis on diestrus-I, neither the follistatin message nor the protein was detectable in the corpora lutea. In conclusion, these results suggest that the follistatin message is present in all the granulosa cells of every developing follicle throughout the estrous cycle, but the follistatin protein appears to be present in only the selected dominant follicles. Accordingly, the possibility that follistatin might be an important regulatory molecule for selection/atresia should be considered.