Oocytes Of Preantral Follicles Research Articles

Metabolic and reproductive parameters in prepubertal gilts after omega-3 supplementation in the diet

Polyunsaturated fatty acids may benefit reproductive performance of female swine. This study evaluated metabolic and reproductive parameters of prepubertal finishing gilts fed with fish oil as a natural source of omega-3 fatty acids (6.88g/d) (n=12) over a period of 45 d. Gilts in the control group were fed soybean oil (n=13). Body weight and backfat were determined at 15-d intervals. Serum levels of leptin, IGF-1, insulin, cholesterol and triglycerides were measured at the beginning (D0) and at the end of the period (D45). Immunolabeling intensity for leptin and its receptor (ObRb) was assessed in oocytes of preantral follicles. Gilts fed omega-3 presented slightly heavier uteri (P=0.09) than control gilts, but there was no effect on body weight and backfat (P>0.05). Cholesterol serum levels tended to be lower at D45 for omega-3 supplemented gilts than for controls (P=0.06). Triglycerides and IGF-1 serum levels were lower at D45 than at D0 for control gilts (P<0.05), but unaltered for supplemented gilts. Insulin levels were unaffected by supplementation (P>0.05), but were greater at D45 than at D0 in both treatments (P<0.05). Immunolabeling for leptin and ObRb in oocytes included in preantral follicles was more intense for supplemented gilts than for control gilts (P<0.05). Omega-3 supplementation was associated with reduced serum cholesterol level and more intense staining for leptin in oocytes of prepubertal gilts, which suggests some involvement on triggering puberty.

Endoplasmic Reticulum-Derived Multilamellar Bodies in Oocytes of Mouse Follicle Cultures under Oxidized Low-Density Lipoprotein Treatment

Objective: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL). Method: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12–48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy. Results: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress. Conclusion: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy.

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: Lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2–4, and 4–6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.

In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone

The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.

Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues

This study assessed the effects of vitrification solutions and equilibration times on morphology of cynomolgus ovarian tissues. Ovarian cortical sections (0.1-0.2 cm thickness) of seven cynomolgus monkeys were randomly allocated to either a control group or one of six vitrification groups. Ovarian tissue sections were vitrified ultra-rapidly by placing them directly into liquid nitrogen using two different vitrification solutions (VSEGP: 5.64 mol/l ethylene glycol+5% (w/v) polyvinylpyrrolidone+0.5 mol/l sucrose; and VSED: 3.22 mol/l ethylene glycol+2.56 mol/l dimethylsulphoxide+0.5 mol/l sucrose) after three different exposure times (5-20 min). After warming, follicle morphology was analysed using light and transmission electron microscopy. The proportion of morphologically normal follicles vitrified using VSED after a 5-min exposure was lower (P<0.05) than those vitrified by other conditions. The proportion of normally structured mitochondria in oocytes of preantral follicles vitrified after a 5-min exposure to VSED (56%) was lower (P<0.01) than those vitrified by other conditions (78-88%). Following tissue vitrification with VSED, the surface ratio of lysosome was increased compared with non-vitrified oocytes (1.64% versus 1.11%; P<0.05). These results indicate that VSEGP can support the morphology of vitrified preantral follicles and oocytes.

Immunohistochemical localization of growth factors after cryopreservation and 3 weeks' xenotransplantation of human ovarian tissue

To compare early follicular growth after fresh and frozen-thawed human ovarian tissue xenografting and to investigate whether expression of c-kit, kit ligand (KL), and growth differentiation factor-9 (GDF-9) is maintained after freezing and xenografting of human ovarian tissue. Prospective experimental study. Gynecology research unit in a university hospital. Ten nude (Swiss nu/nu) 6-week-old female mice. The ovarian biopsy samples were obtained from six women, aged 20 to 30 years. Fresh and frozen-thawed ovarian fragments intraperitoneally grafted into nude mice for 3 weeks. Histologic analysis and immunohistochemical evaluation of c-kit, KL, and GDF-9 expression before and after grafting. The integrity and proportion of growing follicles increased similarly in both fresh (64%) and frozen-thawed (59%) xenografts compared with non-grafted tissue (fresh: 40%; frozen-thawed: 21%). Both C-kit and KL staining were detected in the oocytes and granulosa cells of preantral follicles, and GDF-9 expression was observed in the oocytes of preantral follicles in all groups. Freezing does not appear to have a major impact on early follicular growth after transplantation. This study shows, for the first time, expression of c-kit, KL, and GDF-9 in human preantral follicles after ovarian tissue cryopreservation and xenotransplantation.

Cryopreservation of swine ovarian tissue: Effect of different cryoprotectants on the structural preservation of preantral follicle oocytes

The present study aimed to test different cryoprotectants on cryopreservation of pig ovarian tissue. Pig ovaries ( n = 3) were collected at a local slaughterhouse. From each ovary, ten cortex samples were taken. One was immediately fixed (control) and another placed in short-term tissue incubation (STTI control). The other 8 samples were cryopreserved, in pairs, using 4 different cryoprotectants: dimethyl sulphoxide (Me2SO – 1.5 M), ethylene glycol (EG – 1.5 M), propanediol (PROH – 1.5 M) and glycerol (GLY – 10%), all with 0.4% sucrose. Samples were slow cooled and stored in liquid nitrogen for 7 days. After thawing and cryoprotectant removal, one sample from each treatment was immediately fixed and the other was placed in short-term tissue incubation (STTI) for 2 h and then fixed. Samples were processed for histology and transmission electron microscopy. The percentages of morphologically normal follicles (MNF) in cryopreserved tissue using Me2SO (67.0 ± 4.9), EG (81.8 ± 1.4) and PROH (55.9 ± 9.9) were significantly lower ( P < 0.05) than observed in fresh control tissue (97.7 ± 1.2). When ovarian tissue was cryopreserved with GLY, no morphologically normal follicles could be found (0%). After STTI, PROH showed a significantly lower percentage of MNF when compared with all other treatments and the control. After ultrastructural analysis, follicles cryopreserved with Me2SO and EG showed some small alterations, but no signs of advanced degeneration. Overall, these were similar to follicles from the control group. In conclusion, it is possible to cryopreserve preantral follicles from pig ovarian tissue using Me2SO or EG.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fibroblast growth factor 17 (FGF17) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF17, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF17 protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF17 mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF17 mRNA abundance, and treatment with FGF17 inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF17 is a potential mediator of granulosa cell differentiation.

Expression of transforming growth factor-beta3 (TGF-beta3) in the porcine ovary during the oestrus cycle.

Transforming growth factor-beta (TGF-beta) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-beta1 and TGF-beta2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-beta3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-beta3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-beta3 mRNA is expressed throughout the oestrus cycle. The level of TGF-beta3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-beta3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-beta3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-beta3. Immunostaining of TGF-beta3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-beta3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig.

Characterization of glycoside residues of porcine zona pellucida and ooplasm during follicular development and atresia

The glycoside residues (glycoconjugates, GC) of the zona pellucida (ZP) glycoproteins are important during the first phases of fecundation. Our aim in this work was to determine the lectin affinity pattern of porcine ZP in order to analyze the changes that take place during: (a) preantral folliculogenesis, (b) the follicular atresia process, and (c) antral growth. Several prepubertal and adult pig ovaries and different sized antral follicles were used. Conventional carbohydrate histochemical techniques and peroxidase and digoxigenin (DIG) lectins were used to reveal the acid groups and the glycosidic residues of the ZP. It was seen that the ZP forms in the preantral follicles throughout their growth period. In primordial and primary follicles, ZP in the process of formation showed neutral GC. SBA, RCA-I, MAA, WGA lectins, and AAA after methylation-saponification (MS) were positive in the ZP of primordial and primary follicles. The affinity for SBA, RCA-I, MAA, and WGA increased in the multilaminar-primary follicles and new affinities for UEA-I and LFA were observed. After MS, AAA, SNA, PNA, and SBA reactivity was observed. The ZP of antral follicle oocytes of different sizes showed the same lectin pattern as multilaminar-primary follicles. The oocyte ooplasm and the follicular fluid of large antral follicles showed less affinity for WGA and LFA lectins and less intensive staining with AB (pH 2.5). Atresia did not change the antral or preantral follicle oocyte ZP lectin pattern. In conclusion, the follicles showed substantial changes in their ZP glycosidic composition as they developed, especially, during the change from primary to multilaminar-primary follicles. The ZP glycosidic composition showed no significant change during the growth of antral follicles and follicular atresia in our study.

Regulation of Pcsk6 Expression During the Preantral to Antral Follicle Transition in Mice: Opposing Roles of FSH and Oocytes1

Several secreted products of the TGFbeta superfamily have important roles during follicular development and are produced by both oocytes and somatic cells (granulosa and theca) in the follicle. The proprotein convertases are a family of seven known proteins that process TGFbeta ligands and other secreted products to their mature active form. The present study examined the regulation of steady-state levels of Pcsk6 mRNA, which encodes a convertase protein known to process members of the TGFbeta superfamily, during mouse follicular development. Pcsk6 mRNA and protein were expressed in preantral but not cumulus or mural granulosa cells. Pcsk6 mRNA levels in preantral granulosa cells were not regulated by growing oocytes of preantral follicles, but were elevated by FSH. Furthermore, Pcsk6 mRNA in preantral granulosa cells was potently suppressed by factor(s) secreted by fully grown oocytes from antral follicles, in part through SMAD2/3-mediated pathways. Oocytes acquired the ability to suppress the steady-state levels of Pcsk6 mRNA in granulosa cells during the preantral to antral follicle transition. Suppression of Pcsk6 mRNA by oocytes could reflect a change in the mechanism(s) regulating the activity of members of the TGFbeta superfamily.

Successful ovarian tissue vitrification in mouse, bovine and human

Successful ovarian tissue vitrification in mouse, bovine and human

Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro

There has been controversy over the role of FSH in the regulation of preantral follicle development. LH is a survival and differentiation factor that increases oocyte maturation in FSH-supplemented cultures of mouse preantral follicles. However, little information exists on the action of LH and FSH in the developmental competence of porcine preantral follicle oocytes in vitro. Porcine preantral follicles were cultured for 3 days in the presence or absence of FSH or LH. Oocytes from these follicles were then matured, fertilized in vitro, and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. FSH or combined LH and FSH significantly enhanced follicular growth compared to LH alone or the controls. Combined LH and FSH treatment of preantral follicles significantly increased the percentage (59 +/- 5%) of oocytes competent to undergo cleavage to the two-cell stage after fertilization. A significant effect was seen on oocyte competence to develop from the two-cell to the blastocyst stage (30 +/- 6%) compared to FSH alone treatment (45 +/- 7 and 14 +/- 5%, respectively). The amount of estradiol on days 2 and 3 of culture was significantly higher in follicles cultured with FSH (48.75 +/- 17, 70.5 +/- 14 pg/ml) or combined LH and FSH (63.25 +/- 16, 72.5 +/- 12 pg/ml) than that cultured with the untreated controls (16 +/- 10, 5.66 +/- 4 pg/ml). The results indicated that FSH is essential for the in vitro growth of porcine preantral follicles, estradiol secretion, and for oocytes to acquire competence to resume meiosis and undergo fertilization and embryonic development. LH with FSH treatment of porcine preantral follicles can improve the quality of oocytes by promoting growth and a higher frequency of embryonic development.

Prostaglandin E Receptors

Prostaglandin (PG) E(2) exerts its actions by acting on a group of G-protein-coupled receptors (GPCRs). There are four GPCRs responding to PGE(2) designated subtypes EP1, EP2, EP3, and EP4 and multiple splicing isoforms of the subtype EP3. The EP subtypes exhibit differences in signal transduction, tissue localization, and regulation of expression. This molecular and biochemical heterogeneity of PGE receptors leads to PGE(2) being the most versatile prostanoid. Studies on knock-out mice deficient in each EP subtype have defined PGE(2) actions mediated by each subtype and identified the role each EP subtype plays in various physiological and pathophysiological responses. Here we review recent advances in PGE receptor research.

Ovarian Tissue Banking

ABSTRACT Ovarian tissue cryopreservation is an effective method for protecting against infertility as well as preservating endangered animal species. The technique is particularly sought after as a strategy against ovarian failure caused by aggressive chemotherapy in young women with cancer. There are two uses for cryopreserved ovarian tissues after thawing: grafting and culture. Grafting carries the risk of reintroduction of disease. This article describes the status of ovarian cryopreservation in humans and the other animals and also details the successful birth of a pup from preantral follicle oocytes derived from a mouse cryopreserved ovary followed by in vitro growth, in vitro maturation and in vitro fertilization-embryo transfer (IVF-ET).

Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization

To determine an optimum condition for vitrification of various animal species and to examine the developing capacity into pups of mouse oocytes in preantral follicles originating from cryopreserved ovaries. Experimental animal study. Laboratory environment. Normal (C57BL/6 x DBA2) F1 mice. Vitrification of animal ovaries using polyester sheets as a storage device; collection of oocyte-granulosa cell complexes by enzymatic treatment; and in vitro growth (IVG), in vitro maturation (IVM), and IVF, with embryo transplantation to pseudopregnant mice. Histological analysis of vitrified and warmed ovaries from several animal species; measurement of successful rates in IVG, IVM, and IVF of mouse oocytes in oocyte-granulosa cell complexes collected from the vitrified and warmed ovaries; and achievement of pup birth. The vitrification method used was effective for storage of various animal ovaries. The oocytes enclosed in preantral follicles that were yielded from the vitrified-warmed ovaries preserved the capacity for developing into pups after IVG, IVM, and IVF. The findings indicated that ovarian cryopreservation by vitrification and subsequent IVG, IVM, and IVF are promising methods for restoring young cancer patients' fertility. The practical information presented here is applicable to human ovarian tissues.

The preantral granulosa cell to cumulus cell transition in the mouse ovary: Development of competence to undergo expansion

The transition of preantral to antral follicles is one of the major steps in follicular development, yet little is known about the molecular and functional changes that occur as preantral granulosa cells differentiate into cumulus cells. The cumulus oophorus of large antral follicles undergoes expansion in response to the preovulatory surge of gonadotropins, but preantral granulosa cells do not. The objective of this project was to determine the molecular mechanisms underlying this differential response. Cumulus expansion in vitro requires secretion of cumulus-expansion enabling factors (CEEFs) by the oocyte and stimulation by a ligand, epidermal growth factor (EGF) or follicle-stimulating hormone (FSH). This combined stimulation results in activation of MAPKs (MAPK3/1 (formerly ERK1/2) and MAPK14 (formerly p38)) and increased Has2, Ptgs2, Tnfaip6 and Ptx3 mRNA levels, all of which are required for cumulus expansion. Only fully-grown oocytes from antral follicles were competent to enable expansion and increases in expansion-related transcripts in cumulus cells, whereas growing oocytes of preantral follicles did not. To assess the competence of preantral granulosa cells to generate responses associated with expansion, they were treated with FSH or EGF and co-cultured with fully-grown oocytes secreting CEEFs. MAPKs were activated by EGF in preantral granulosa cells to essentially the same levels as in cumulus cells. Preantral granulosa cells treated with EGF, but not those treated with FSH increased Has2, Ptgs2 and Ptx3 mRNAs to 17–96% of the levels observed in cumulus cells. In contrast, the level of Tnfaip6 mRNA was minimally stimulated in preantral granulosa cells. Therefore, preantral granulosa cells do not undergo expansion for two fundamental reasons. First, the growing oocytes of preantral follicles do not secrete active CEEFs. Second, activation of MAPKs alone in preantral granulosa cells, even in the presence of CEEFs, is not sufficient to increase the expression of essential transcripts, particularly Tnfaip6 mRNA. Thus, preantral granulosa cells differ from cumulus cells in CEEF-dependent processes downstream of the activation of MAPKs.

Effect of growth factors on in vitro development of caprine preantral follicle oocytes

The objective of this work was to examine the effect of various growth factors including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), either individually or in association, in the presence of follicle stimulating hormone (FSH) on the in vitro growth and viability of caprine preantral follicle oocytes. Preantral follicles were disassociated enzymatically and mechanically from prepuberal caprine ovaries after the animals were anesthetically ovariectomized. In experiment, caprine preantral follicles in groups 1–4 were cultured in growth culture medium, growth culture medium + EGF, growth culture medium + IGF-I and growth culture medium + IGF-I + EGF, respectively, for 9 days. The results indicated that EGF (50 mg/l) increased the survival rate of oocytes, but decreased the growth rate of oocytes; IGF-I (100 mg/l) effectively maintained the survival of oocytes and stimulated their growth; IGF-I (100 mg/l) and EGF (50 mg/l) in combination produced a higher effect on both of the survival and the growth rate of oocytes than IGF-I or EGF alone. Conclusively, the growth factors can effectively maintain the survival of caprine preantral follicle oocytes and regulated their growth in culture. EGF and IGF-I in association could synergically meliorate the culture system of caprine preantral follicle oocytes.

Expression of Sox8, Sf1, Gata4, Wt1, Dax1, and Fog2 in the mouse ovarian follicle: Implications for the regulation of Amh expression

Anti-Mullerian hormone (Amh) is expressed in the granulosa cells of growing and preovulatory follicles in the mouse ovary where it acts to decrease responsiveness to follicle stimulating hormone (FSH) and plays a role in inhibiting primordial follicle recruitment. Recently, co-culture of isolated oocytes and granulosa cells has demonstrated that Amh expression is up-regulated in the presence of oocytes and, at preantral stages, this effect is dependent upon close contact. In Sertoli cells, Amh expression is regulated by several transcription factors including SOX9, SF1, GATA4, WT1, and DAX1, which, with the exception of SOX9, are also expressed in granulosa cells where GATA4 is known to up-regulate Amh expression antagonised by FOG2. Here, we demonstrate that Sox8, which is closely related to Sox9 and encodes the protein SOX8 which can transactivate Amh, is expressed in the postnatal mouse ovarian follicle but is not co-expressed with Amh in the granulosa cells of preantral follicles. Sox8 expression was found only in the oocytes of preantral follicles and in the oocytes, cumulus cells, and mural granulosa cells of preovulatory follicles. Also, increased expression of Amh in granulosa cells co-cultured with oocytes was not associated with increased mRNA levels of the transcription factors Sf1, Gata4, Wt1, Dax1, or Fog2. These findings reveal Sox8 expression in the ovarian follicle and show that oocyte regulation of Amh expression is not due to oocyte regulation of Sf1, Gata4, Wt1, Dax1, or Fog2 expression in granulosa cells.

Small nuclear RING finger protein expression during gonad development: regulation by gonadotropins and estrogen in the postnatal ovary.

Small nuclear RING finger protein (SNURF/RNF4) is a steroid receptor coregulator that is down-regulated in testicular germ cell cancer. In this work, we examined SNURF expression during murine fetal gonad development and postnatal ovarian folliculogenesis by in situ hybridization and immunohistochemical staining. SNURF mRNA was detectable in gonads of both sexes from embryonic 10.5 days post conception onward. SNURF protein localized to gonocytes and somatic Leydig and Sertoli cells of fetal testis and in oogonia and supporting cells of fetal ovary. In murine postnatal ovary, SNURF mRNA and protein were expressed throughout folliculogenesis, peaking in the oocytes of preantral follicles. Lower amounts of SNURF mRNA and protein were also present in granulosa cells of secondary, antral, and preovulatory follicles and in luteal glands. Exposure of immature female mice and rats to gonadotropin from pregnant mare serum and human chorionic gonadotropin did not change dramatically SNURF mRNA levels in ovary. SNURF mRNA expression was increased in ovaries of immature mice treated with diethylstilbestrol, an effect that was blocked by the pure antiestrogen ICI 182,780. SNURF protein was constitutively expressed in oocytes of hypophysectomized rats, and its content was augmented by estradiol in granulosa cells. In granulosa cell culture, SNURF mRNA accumulation was transiently increased by treatment with the LH agonists phorbol myristate and forskolin at 4 h after treatment and at 48 h in differentiated cells expressing markers of the preovulatory phenotype. These results suggest a role for SNURF in fetal germ cell development as well as in oocyte and granulosa cell maturation in an estrogen- and gonadotropin-regulated fashion.