Immunohistochemical Localization of Taurine in the Rat Ovary, Oviduct, and Uterus

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133–1142, 2001)

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Gene expression and protein localisation for activin-A, follistatin and activin receptors in goat ovaries

Journal of Endocrinology ◽

10.1677/joe.1.05756 ◽

2004 ◽

Vol 183 (2) ◽

pp. 405-415 ◽

Cited By ~ 28

Author(s):

J R V Silva ◽

R van den Hurk ◽

H T A van Tol ◽

B A J Roelen ◽

J R Figueiredo

Keyword(s):

Mrna Expression ◽

Granulosa Cells ◽

Activin A ◽

Surface Epithelium ◽

Corpora Lutea ◽

Rt Pcr ◽

Primordial Follicles ◽

Antral Follicles ◽

Theca Cells ◽

Activin Receptors

We studied the protein and mRNA expression of activin-A, follistatin and activin receptors in goat ovaries to find evidence of their possible role in ovarian activity, particularly in the various stages of follicle development. Ovaries of cyclic goats were collected and then either fixed in paraformaldehyde for immunohistochemical localisation of activin-A, follistatin, activin receptors IIA/B (ActR-IIA/B) and IA (ActR-IA) proteins or used to obtain samples to demonstrate mRNA expression of activin-A (βA subunit), follistatin, ActR-IIA, -IIB, -IA and -IB, using RT-PCR. For this latter goal, primordial, primary and secondary follicles were isolated mechanically, washed to remove the stromal cells and then used for RT-PCR. In addition, oocytes, cumulus, mural granulosa and theca cells from small (<3 mm) and large (3–6 mm) antral follicles, luteal cells and surface epithelium were collected to study mRNA expression. Activin-A and follistatin proteins were found in oocytes of all follicle classes, granulosa cells from the primary follicle stage onwards, theca cells of antral follicles, corpora lutea and ovarian surface epithelium. In antral follicles, these proteins were detected both in cumulus and mural granulosa cells. ActR-IIA/B protein was found at the same follicular sites, and also in granulosa cells of primordial follicles onward. The localisation of ActR-IA corresponded with that of ActR-IIA/B, but the former protein was absent in the theca of large antral follicles. The mRNAs for activin-A (βA subunit), follistatin, and ActR-IIA, -IIB, -IA and -IB were detected at all follicular and cellular types studied, except that ActR-IIB was not found in follicles that had not developed an antrum yet. In conclusion, in goat ovaries, transcripts of activin-A (βA subunit), its receptors and its binding protein follistatin are expressed and their proteins formed at all follicular stages and in corpora lutea. These findings indicate a role of activin-A in the local regulatory system during the entire follicular development and during luteal activity.

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107 Localization of Kisspeptin Immunoreactivity in the Cat Ovary on Different Reproductive Stages

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab107 ◽

2018 ◽

Vol 30 (1) ◽

pp. 193

Author(s):

P. Tanyapanyachon ◽

O. Amelkina ◽

K. Chatdarong

Keyword(s):

Corpus Luteum ◽

Granulosa Cells ◽

Ovarian Surface Epithelium ◽

Domestic Cat ◽

Primary Antibody ◽

Surface Epithelium ◽

Domestic Cats ◽

Luteal Cells ◽

Theca Cells ◽

Steroidogenic Cells

Kisspeptin (Kp) is considered one of the main regulators of the reproductive axis, exerting its effects via stimulating GnRH expression in the hypothalamus. Apart from its central localization in the hypothalamus, the presence of Kp has been reported in the ovary, with possible local function. To date, very little is known about the ovarian Kp in the domestic cat. Therefore, our aim was to investigate the presence and localization of Kp at different reproductive stages in domestic cat ovaries. Twenty ovaries were collected from free-ranging domestic cats (body weight 2.7–4.5 kg) after routine ovariohysterectomy. Reproductive stages were classified by ovarian gross morphology, vaginal cytology, and blood progesterone level. Ovarian samples were grouped into inactive (n = 6), follicular (n = 8), and luteal stages (n = 6). Tissues were fixed in 4% paraformaldehyde and processed routinely. Immunohistochemistry was performed using polyclonal rabbit Kp-10 primary antibody (AB9754; Millipore, Billerica, MA, USA) at 1:500 at 4°C overnight. Immunoreactive cells were identified by avidin-biotin-peroxidase system. Rat hypothalamic tissue was used as a positive control. Primary antibody was substituted with PBS and normal rabbit IgG as the negative and isotypic negative controls, respectively. In addition, primary antibody was incubated with metastin overnight and applied for preabsorption test. Negative, isotypic negative, and preabsorption tests showed no staining. Immunoreactive Kp was detected in the ovaries of all reproductive stages with no obvious changes in localization or intensity of staining between stages. Kisspeptin was present in the cytoplasm of oocytes, granulosa cells, and theca cells of preantral (primordial, primary, and secondary) follicles and antral follicles. Interestingly, in most follicles, Kp staining was more prominent in theca cells and oocytes compared with granulosa cells. In corpus luteum, Kp was localised in the cytoplasm of luteal cells, with more intense staining on the periphery of corpus luteum compared with the middle in 3 luteal samples, whereas the rest of the samples demonstrated homogeneous staining distribution. Apart from oocytes and steroidogenic cells, Kp was also present in the cytoplasm of cells of the ovarian surface epithelium. Our study for the first time demonstrated the presence and localization of Kp in the ovary of the domestic cats. The localization of Kp in the cat oocyte is similar to previous reports on hamsters and dogs, indicating a possible function in oocyte development. The staining in steroidogenic cells, mainly theca cells and luteal cells, is in good agreement with studies on hamsters, rats, humans, and marmosets, suggesting the possible local involvement of Kp in steroidogenesis. In addition, Kp staining in the ovarian surface epithelium suggests a possible role in the ovarian remodeling after ovulatory defects, as reported in humans and marmosets. This research was funded by the RGJ PhD program PHD/01882556; RG 7/2559.

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Heme Oxygenase in the Rat Ovary: Immunohistochemical Localization and Possible Role in Steroidogenesis

Experimental Biology and Medicine ◽

10.1177/153537020322800108 ◽

2003 ◽

Vol 228 (1) ◽

pp. 59-63 ◽

Cited By ~ 9

Author(s):

Iulia C. Alexandreanu ◽

David M. Lawson

Keyword(s):

Heme Oxygenase ◽

Granulosa Cells ◽

Corpora Lutea ◽

Sprague Dawley ◽

Hormone Production ◽

Similar Treatment ◽

Theca Cells ◽

Ovarian Stroma ◽

Rat Ovary

The objectives of this study were to determine if heme oxygenase (HO), which catalyzes the degradation of heme and the formation of carbon monoxide (CO), is localized in the rat ovary and, if so, to determine if hemin (a substrate for HO) or chromium mesoporphyrin (CrMP, an inhibitor of HO), alter basal or gonadotropin-induced steroidogenesis. The hypothesis was that CO produced endogenously by HO suppresses steroid hormone production by the ovary similar to the action of nitric oxide. For the histological localization of HO, sections of ovaries obtained from mature Holtzman Sprague-Dawley rats were immunostained for two of the HO isoforms, HO-1 and HO-2. Theca cells and granulosa cells of follicles and luteal cells stained for HO-1, whereas the ovarian stroma showed a low intensity of staining. Theca, granulosa cells, and corpora lutea as well as the ovarian stroma exhibited HO-2 staining. HO-2 immunostaining appeared more intense for theca cells than granulosa cells. In the study of steroidogenesis, three daily injections of hemin stimulated basal- and gonadotropin-induced androstenedione and estradiol secretion from ovaries of pregnant mare serum gonadotropin-treated immature rats in vitro, but had no effect on progesterone production. A similar treatment with CrMP suppressed basal- and gonadotropin-induced secretion of progesterone and androstenedione, but had no effect on estradiol production. These data, taken together, show the existence of HO in the rat ovary and suggest a possible stimulatory role of endogenous CO in the production of ovarian steroids.

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The Kit ligand/c-Kit receptor system in goat ovaries: gene expression and protein localization

Zygote ◽

10.1017/s0967199406003832 ◽

2006 ◽

Vol 14 (4) ◽

pp. 317-328 ◽

Cited By ~ 25

Author(s):

J.R.V. Silva ◽

R. van den Hurk ◽

H.T.A. van Tol ◽

B.A.J. Roelen ◽

J.R. Figueiredo

Keyword(s):

Granulosa Cells ◽

Protein Localization ◽

Primordial Follicle ◽

Surface Epithelium ◽

Corpora Lutea ◽

Antral Follicles ◽

Theca Cells ◽

Kit Ligand ◽

Medullary Tissue ◽

Sites Of Action

SummaryRelatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.

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Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

Reproduction ◽

10.1530/rep-14-0436 ◽

2015 ◽

Vol 150 (1) ◽

pp. 11-20 ◽

Cited By ~ 7

Author(s):

Tonny Studsgaard Petersen ◽

Martin Stahlhut ◽

Claus Yding Andersen

Keyword(s):

Primordial Follicle ◽

Neonatal Rat ◽

Intracellular Camp ◽

Corpora Lutea ◽

Dependent Manner ◽

Primordial Follicles ◽

Primordial Follicle Activation ◽

Rat Ovary ◽

Follicle Differentiation ◽

Follicle Activation

Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15–26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation.

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Immunolocalisation of oestrogen receptor beta in human tissues

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0240145 ◽

2000 ◽

Vol 24 (1) ◽

pp. 145-155 ◽

Cited By ~ 333

Author(s):

AH Taylor ◽

F Al-Azzawi

Keyword(s):

Epithelial Cells ◽

Granulosa Cells ◽

Oestrogen Receptor ◽

Cell Types ◽

Corpora Lutea ◽

Human Tissues ◽

Cell Nuclei ◽

Reverse Transcription Pcr ◽

Wide Range ◽

Protein Receptors

Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.

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11β-Hydroxysteroid dehydrogenase expression and activities in bovine granulosa cells and corpora lutea implicate corticosteroids in bovine ovarian physiology

Journal of Endocrinology ◽

10.1677/joe.1.07025 ◽

2007 ◽

Vol 193 (2) ◽

pp. 299-310 ◽

Cited By ~ 15

Author(s):

L M Thurston ◽

D R E Abayasekara ◽

A E Michael

Keyword(s):

Molecular Mass ◽

Granulosa Cells ◽

Luteal Phase ◽

Hydroxysteroid Dehydrogenase ◽

Follicular Phase ◽

Corpora Lutea ◽

Luteal Cells ◽

Antral Follicles ◽

Follicle Diameter ◽

Dependent Type

Cortisol–cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) enzyme and the oxidative NAD+-dependent type 2 11βHSD (11βHSD2). This study related the expression of 11βHSD1 and 11βHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11β-dehydrogenase (11β-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of < 4, 4–8, > 8 and > 12 mm in diameter in either the follicular or luteal phase of the ovarian cycle. Luteal cells were obtained from corpora lutea (CL) in the early non-pregnant luteal phase. Enzyme expression was assessed by reverse transcription-PCR and western blotting, while enzyme activities were measured over 1 h in cell homogenates using radiometric conversion assays with 100 nM [3H]cortisone or [3H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11βHSD2 and NAD+-dependent oxidation of cortisol predominated in granulosa cells harvested in the follicular phase. In contrast, in granulosa cells obtained from luteal phase follicles and in bovine luteal cells, expression of 11βHSD1 exceeded that of 11βHSD2 and the major enzyme activity was NADP+-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11βHSD2 and 11βHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11βHSD1 migrated with a molecular mass of 34 kDa, whereas in the dominant follicle, CL and all luteal phase granulosa cells, a second protein band of 68 kDa was consistently detected. In all samples, 11βHSD2 had a molecular mass of 48 kDa, but in large antral follicles (> 8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11βHSD2 is the predominant functional 11βHSD enzyme expressed in follicular phase granulosa cells from growing bovine antral follicles. In contrast, in bovine granulosa cells from dominant or luteal phase follicles, and in bovine luteal cells from early non-pregnant CL, 11βHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP+- and NAD+-dependent 11β-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

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99 GLUCOCORTICOID RECEPTORS ARE EXPRESSED IN OVARIES OF NEWBORN AND ADULT FEMALE HORSES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab99 ◽

2015 ◽

Vol 27 (1) ◽

pp. 142

Author(s):

D. Scarlet ◽

I. Walter ◽

C. Aurich

Keyword(s):

Oocyte Maturation ◽

Granulosa Cells ◽

Glucocorticoid Receptors ◽

Ovarian Tissue ◽

Rna Extraction ◽

Cumulus Cells ◽

Oestrous Cycle ◽

Luteal Function ◽

Luteal Cells ◽

Theca Cells

In contrast to other domestic animal species, in vitro maturation (IVM) of oocytes in the horse is still not successful. Oocytes for IVM are obtained either from slaughterhouse ovaries or via ovum pick-up from living mares. Both situations may be associated with a stress-induced glucocorticoid release. So far, neither an involvement of glucocorticoids in follicle and oocyte maturation nor the presence of glucocorticoid receptors (GCR) in ovarian tissue has been investigated in the horse. We hypothesised that GCR are expressed in equine ovarian tissue independent of the animal's age and stage of the oestrous cycle. Ovaries (n = 40) were collected from killed newborn female foals (n = 10) and killed or slaughtered adult mares (n = 10). For assessment of GCR mRNA expression, ovarian samples were fixed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zoeterwoude, the Netherlands) and stored at –80°C. Various cell populations were isolated using laser capture microdissection on cryosections. After RNA extraction, samples were analysed by qualitative RT-PCR and real time-PCR. For analysis of GCR protein, tissue was fixed in Bouin's solution and histological slides immunostained using a monoclonal antibody for GCR (Ab2768, Abcam, Cambridge, UK), followed by visualisation with diaminobenzidine. One tertiary follicle per slide (40×; light microscopy) was analysed and percentages of cells staining positive for GCR calculated. Statistical analysis was done with the SPSS Statistics 21 software (SPSS Inc., Chicago, IL, USA). Expression of mRNA for GCR was detected in oocytes, cumulus cells, granulosa, and theca cells, independent of age and stage of the oestrous cycle. In both neonates and adults, nuclei of the oocytes and cumulus cells stained positive for GCR regardless of stage of folliculogenesis. Also, GCR were constantly expressed in granulosa cells from both preantral and antral follicles. Percentage of granulosa cells staining positive for GCR (adult: 73.6 ± 3.2, fillies: 72.4 ± 1.9%) was higher (P < 0.001) than of theca cells (adult: 56.8 ± 3.9, fillies: 57.2 ± 1.9%), but not affected by age. GCR were lacking in ovarian stroma of adults but not of neonates. In periovulatory follicles from adult mares, GCR were abundant in developing luteal cells. GCR were also detected in the nuclei of luteal cells in corpora haemorrhagica and corpora lutea. Follicular atresia was associated with a decrease of GCR independent of cell type and age. This study describes for the first time the expression of GCR in horse ovaries, which are present independent of age of the animal, stage of folliculogenesis, and oestrous cycle stage. Results suggest that glucocorticoids are involved in follicular and oocyte maturation, ovulation, and luteal function in the horse. Presence of GCR in the ovaries of newborn horses suggests a role of glucocorticoids in ovarian tissue maturation. Nevertheless, detrimental effects of excess glucocorticoid secretion due to stress on follicular development, oocyte maturation, and luteal function cannot be excluded in the mare.

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Parallel effects of Pulsatilla and exogenous leutocyclin on the rat ovary, uterus and thyroid

British Homeopathic Journal ◽

10.1016/s0007-0785(78)80053-2 ◽

1978 ◽

Vol 67 (04) ◽

pp. 265-275 ◽

Cited By ~ 6

Author(s):

Suresh Prasad ◽

K. Chandrasekhar

Keyword(s):

Epithelial Cells ◽

Comparative Study ◽

Ovarian Follicles ◽

Mitotic Division ◽

Albino Rats ◽

Corpora Lutea ◽

Luminal Epithelium ◽

Simultaneous Decrease ◽

Rat Ovary

SummaryA comparative study of Pulsatilla (a homœopathic medicine) and leutocyclin was undertaken in female albino rats showing regular oestrus cycles. Pulsatilla in the 30th and 200th potencies was administered orally, and leutocyclin was injected in doses of 0·125 mg and 1·25 mg. 0·25 ml/dose of 90 per cent. alcohol was used as the vehicle. Pulsatilla 30 and 200 and leutocyclin at a dose of 1·25 mg markedly reduced the weight of the ovary. The higher dose of leutocyclin (1·25 mg) and both the attenuations of Pulsatilla increased the process of atresia in ovarian follicles. But a simultaneous decrease in the number and diameters of the corpora lutea was observed.Administration of Pulsatilla and leutocyclin resulted in reduction in the weight of the uterus and the height of the endometrium and luminal epithelium. There was also a reduction in the height of epithelial cells in the thyroid. Both Pulsatilla and leutocyclin enhanced mitotic division in the uterine stroma.The results indicate that leutocyclin in doses of between 0·125 mg and 1·25 mg may be used to produce similar changes in the ovary, uterus and thyroid of the rat as Pulsatilla 30 and 200.

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Fibroblast Growth Factor-9, a Local Regulator of Ovarian Function

Endocrinology ◽

10.1210/en.2006-1668 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3711-3721 ◽

Cited By ~ 28

Author(s):

Ann E. Drummond ◽

Marianne Tellbach ◽

Mitzi Dyson ◽

Jock K. Findlay

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Granulosa Cells ◽

Ovarian Function ◽

Corpora Lutea ◽

Mrna Levels ◽

Fibroblast Growth ◽

Immature Rat ◽

Progesterone Production ◽

Rat Ovary

Fibroblast growth factor 9 (FGF9) is widely expressed in embryos and fetuses and has been shown to be involved in male sex determination, testicular cord formation, and Sertoli cell differentiation. Given its male gender bias, the ovary has not been reported to express FGF9, nor has a role in ovarian function been explored. We report here that FGF9 mRNA and protein are present in the rat ovary and provide evidence that supports a role for FGF9 in ovarian progesterone production. FGF9 mRNA levels as determined by real-time PCR were high in 4-d-old rat ovaries, thereafter declining and stabilizing at levels approximately 30% of d 4 levels at d 12–25. Levels of FGF9 mRNA in the ovary were significantly higher than that present in adult testis, at all ages studied. The FGF9 receptors FGFR2 and FGFR3 mRNAs were present in postnatal and immature rat ovary and appeared to be constitutively expressed. FGF9 protein was localized to theca, stromal cells, and corpora lutea and FGFR2 and FGFR3 proteins to granulosa cells, theca cells, oocytes, and corpora lutea, by immunohistochemistry. Follicular differentiation induced by gonadotropin treatment reduced the expression of FGF9 mRNA by immature rat ovaries, whereas the estrogen-stimulated development of large preantral follicles had no significant effect. In vitro, FGF9 stimulated progesterone production by granulosa cells beyond that elicited by a maximally stimulating dose of FSH. When the granulosa cells were pretreated with FSH to induce LH receptors, FGF9 was found not to be as potent as LH in stimulating progesterone production, nor did it enhance LH-stimulated production. The combined treatments of FSH/FGF9 and FSH/LH, however, were most effective at stimulating progesterone production by these differentiated granulosa cells. Analyses of steroidogenic regulatory proteins indicate that steroidogenic acute regulatory protein and P450 side chain cleavage mRNA levels were enhanced by FGF9, providing a mechanism of action for the increased progesterone synthesis. In summary, the data are consistent with a paracrine role for FGF9 in the ovary.

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