Oocytes Of Primordial Follicles Research Articles

Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles

The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.

Growth of Primordial Oocytes in Neonatal and Adult Mammals

Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.

95 IN VITRO MATURATION AND FERTILIZATION OF OOCYTES FROM OVARIAN TISSUES CRYOPRESERVED AND XENOGRAFTED INTO NUDE MICE

Primordial follicles act as stores of ovarian follicles and are potential sources of oocytes for medical, agricultural, and zoological purposes. Ovarian xenografting seems to be advantageous for maturing the oocytes in primordial follicles (primordial oocytes) and useful for the conservation and reproduction of domestic or endangered animals. We have generated viable embryos from porcine primordial oocytes xenografted into nude mice (Kaneko et al. 2006 Reproduction). Xenografting of ovarian tissues after cryopreservation would be a very powerful tool for this purpose. Recently, Moniruzzaman et al. 2009 Theriogenology) reported that follicles were able to develop to the pre-antral stage in ovarian tissues after cryopreservation and xenografting, but that oocytes were not obtainable from them. In the present study, we vitrified the tissue after different immersion periods in a cryoprotectant, ethylene glycol (EG), and evaluated the possibility of oocyte collection, and also their maturation and fertilization abilities. Ovarian tissue from piglets approximately 20 days old was minced into cubes of about 1.0 to 2.0 mm. After equilibration in 4% EG in IVC-PyrLac (Kikuchi et al. 2002 Biol. Reprod.) as a base solution (BS) for 15 min, they were immersed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BS) for 45 s or 7 min (45-s and 7-min immersion groups, respectively), then dropped with about 4 μL of vitrification solution into liquid nitrogen (LN2). After storage in LN2, microdroplets were transferred in warming solution (0.4 M trehalose in BS) at 37°C for 2 min, then consecutively transferred for 2-min periods into 0.2 M, 0.1 M, or 0.05 M trehalose in BS.As described previously (Kaneko et al. 2006 Reproduction), 20 to 30 pieces of tissue were grafted into kidney capsules of ovariectomized nude mice. The host mice were treated with porcine FSH (62.5 U mL-1 in osmotic pomp) for 12 days before assessment of the survival of the ovarian grafts. When antral follicles were evident in the grafts, oocytes were collected using a surgical blade. They were then matured and fertilized in vitro (Kikuchi et al. 2002 Biol. Reprod.). Ovaries containing antral follicles were obtained between 62 and 125 days after grafting from 6 out of 12 mice in the both 45-s and 7-min groups. When a total of 39 and 49 fully grown oocytes, respectively, had been collected from these groups and cultured, the maturation rates calculated on the basis of 1st polar body extrusion were 18% (7/39) and 33% (16/49), respectively. The corresponding rates for sperm-penetrated oocytes were 83% (5/6) and 88% (14/16), respectively. All the oocytes formed male and female pronuclei at 10 h after insemination. The rates in the 2 immersion groups did not differ significantly by chi-square test, with or without Yates’ correction. In conclusion, fully grown porcine oocytes can be collected from primordial follicles that have been cryopreserved and xenografted into nude mice. The period of immersion before vitrification may not affect oocyte maturation or fertilization ability.

Knockdown of FOXO3 induces primordial oocyte activation in pigs

Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10- to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94+/-2%) primordial oocyte nuclei, and in infant pigs, 42+/-7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.

Oocyte-Specific Deletion of Pten in Mice Reveals a Stage-Specific Function of PTEN/PI3K Signaling in Oocytes in Controlling Follicular Activation

Immature ovarian primordial follicles are essential for maintenance of the reproductive lifespan of female mammals. Recently, it was found that overactivation of the phosphatidylinositol 3-kinase (PI3K) signaling in oocytes of primordial follicles by an oocyte-specific deletion of Pten (phosphatase and tensin homolog deleted on chromosome ten), the gene encoding PI3K negative regulator PTEN, results in premature activation of the entire pool of primordial follicles, indicating that activation of the PI3K pathway in oocytes is important for control of follicular activation. To investigate whether PI3K signaling in oocytes of primary and further developed follicles also plays a role at later stages in follicular development and ovulation, we conditionally deleted the Pten gene from oocytes of primary and further developed follicles by using transgenic mice expressing zona pellucida 3 (Zp3) promoter-mediated Cre recombinase. Our results show that Pten was efficiently deleted from oocytes of primary and further developed follicles, as indicated by the elevated phosphorylation of the major PI3K downstream component Akt. However, follicular development was not altered and oocyte maturation was also normal, which led to normal fertility with unaltered litter size in the mutant mice. Our data indicate that properly controlled PTEN/PI3K-Akt signaling in oocytes is essential for control of the development of primordial follicles whereas overactivation of PI3K signaling in oocytes does not appear to affect the development of growing follicles. This suggests that there is a stage-specific function of PTEN/PI3K signaling in mouse oocytes that controls follicular activation.

Neurotrophin NT3 promotes ovarian primordial to primary follicle transition

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100-250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrphostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition.

Testosterone Induces Porcine Primordial Follicle Development In Vitro.

Mammalian ovaries contain a large stock of oocytes enclosed in primordial follicles. A limited number of primordial follicles start to develop, while the remaining large numbers of them are quiescent. The mechanisms controlling the initiation of oocyte growth and primordial follicle development remain to be elucidated. Recent studies have shown that testosterone regulates early stages of folliculogenesis and follicle recruitment in vivo and in vitro. The objective of this study was to examine the effect of testosterone on initiation of primordial follicle development in cultured porcine ovarian tissues and subsequent xenotransplantation. Small pieces (1 mm × 1 mm × 1 mm) of ovarian cortex containing primordial follicles were dissected from ovaries of prepubertal pigs, and cultured in α-minimum essential medium supplemented with bovine plasma, insulin-transferrin-selenium, 2-mercaptoethanol, sodium pyruvate and different concentrations of testosterone (0, 10-7, 10-6, 10-5 and 10-4 M) or estradiol-17β (0, 10-7, 10-6 and 10-5 M) for 7 days. They were then fixed to examine the development of primordial follicles histologically. Ovarian tissues before culture contained primordial follicles (85 ± 6%) and small proportion of degenerated follicles (15 ± 6%). After 7 days of culture without hormone (control), no primordial follicle developed to the intermediate (early primary) or primary stage, and 56 ± 2% follicles were degenerated. On the other hand, in the tissues cultured with 10-6 M testosterone, higher percentages of intermediate follicles and small proportion of primary follicles were observed (17 ± 4% and 1 ± 1%, respectively). Percentages of degenerated follicles were significantly lower in 10-6 M (24 ± 1%) and 10-5 M (34 ± 1%) testosterone groups compared to the control. Estradiol-17β at any concentrations did not affect follicle development, although 10-6 M estradiol-17β diminished the percentage of degenerated follicles (40 ± 4%) compared to the control (61 ± 6%). Addition of an androgen receptor antagonist, cyproterone acetate (10-7 and 10-6 M) inhibited the stimulatory effect of testosterone (10-6 M) on primordial follicle development, suggesting the receptor-mediated action of testosterone. We next examined the presence of the androgen receptor in primordial follicles. Immunohistological examination of ovarian cortical sections revealed that androgen receptor signals were localized in oocytes in primordial follicles. In addition, in the Western blot analysis, anti-androgen receptor antibody recognized a band around 100 kDa in isolated oocytes from primordial follicles. Finally, the effect of testosterone on primordial follicle development was confirmed by xenotransplantation. Ovarian cortical pieces cultured for 7 days with and without 10-6 M testosterone were transplanted under the kidney capsule of female severe combined immune deficient mice for two months. Histological analysis of testosterone-treated xenografts showed that higher percentages of follicles developed to the primary, secondary and antral follicles compared to the control grafts in which no follicular development was observed. In conclusion, testosterone induces porcine primordial follicle development in vitro. (poster)

Neurotrophin Nt3 Promotes Ovarian Primordial to Primary Follicle Transition.

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100-250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrphostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition.

Addition of a Phosphatase Inhibitor Prompts Growth Initiation of Isolated Primordial Follicle Oocytes in Culture.

Addition of a Phosphatase Inhibitor Prompts Growth Initiation of Isolated Primordial Follicle Oocytes in Culture.

The GnRH analogue triptorelin confers ovarian radio-protection to adult female rats

There is a controversy regarding the effects of the analogues of the gonadotrophin-releasing hormone (GnRH) in radiotherapy. This has led us to study the possible radio-protection of the ovarian function of a GnRH agonist analogue (GnRHa), triptorelin, in adult, female rats ( Rattus norvegicus sp.). The effects of the X-irradiation on the oocytes of ovarian primordial follicles, with and without GnRHa treatment, were compared, directly in the female rats (F 0) with reproductive parameters, and in the somatic cells of the resulting foetuses (F 1) with cytogenetical parameters. In order to do this, the ovaries and uteri from 82 females were extracted for the reproductive analysis and 236 foetuses were obtained for cytogenetical analysis. The cytogenetical study was based on the data from 22,151 metaphases analysed. The cytogenetical parameters analysed to assess the existence of chromosomal instability were the number of aberrant metaphases (2234) and the number (2854) and type of structural chromosomal aberrations, including gaps and breaks. Concerning the reproductive analysis of the ovaries and the uteri, the parameters analysed were the number of corpora lutea, implantations, implantation losses and foetuses. Triptorelin confers radio-protection of the ovaries in front of chromosomal instability, which is different, with respect to the single and fractioned dose. The cytogenetical analysis shows a general decrease in most of the parameters of the triptorelin-treated groups, with respect to their controls, and some of these differences were considered to be statistically significant. The reproductive analysis indicates that there is also radio-protection by the agonist, although minor to the cytogenetical one. Only some of the analysed parameters show a statistically significant decrease in the triptorelin-treated groups.

FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles.However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Developmental regulation of the expression of the transferrin receptor and Ki67 in oocytes of the baboon fetal ovary by estrogen

We previously showed that estrogen regulates baboon fetal ovarian follicle development and oocyte integrity. Because iron incorporated into cells by the transferrin receptor is essential for cell/nuclear function, we determined whether fetal oocyte expression of transferrin receptor and the nuclear protein Ki67 were developmentally regulated by estrogen and associated with DNA integrity/fragmentation. Transferrin-receptor expression was minimal at midgestation and abundant in late gestation and localized to the cytoplasm and surface of oocytes of primordial follicles. Expression of transferrin receptor, however, was negligible in oocytes in fetuses in which serum estradiol-17beta levels were suppressed (>95%) by daily maternal treatment between mid- and late gestation with the aromatase inhibitor letrozole and partially restored by treatment with letrozole and estradiol benzoate. Ki67 was localized to pregranulosa and germ cells at midgestation and throughout the oocyte nucleus in late gestation in estrogen-replete fetuses. In contrast, in estrogen-suppressed fetuses, Ki67 was localized to a limited number of foci around the oocyte nucleus. Apoptosis detected in pregranulosa and germ cells at midgestation was not observed in late gestation in estrogen-replete/-suppressed fetuses. We conclude that estrogen regulates fetal oocyte transferrin-receptor expression and that inhibition of receptor development is associated with alterations in Ki67 expression by the oocyte but not apoptosis. Collectively, these results and our previous studies further define the essential role of estrogen in regulating development of follicles comprised of healthy oocytes by the baboon fetal ovary.

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.

The in vitro growth and maturation of follicles

The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell-cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.

Suppression of Notch Signaling in the Neonatal Mouse Ovary Decreases Primordial Follicle Formation

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.

Hormone-sensitive Lipase Expression and IHC Localization in the Rat Ovary, Oviduct, and Uterus

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.

Regulation of Expression of Microvillus Membrane Proteins by Estrogen in Baboon Fetal Ovarian Oocytes1

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.

415. Increased expression of an androgen receptor regulated gene, kit ligand, in polycystic ovaries

Polycystic ovaries (PCO) are induced by pathological or pharmacological female androgen excess, but the role of the androgen receptor (AR) in the pathogenesis of PCO is unknown. We therefore tested the hypothesis that PCO have increased expression of AR or kit ligand (KITL), a cytokine that was recently identified as a candidate AR-regulated gene in the ovary (1). Immunohistochemical analysis of AR and KITL expression was performed on archival paraffin-embedded sections of 8 morphologically normal and 8 polycystic ovaries from women under the age of 40 years. Stained sections were scanned with a NanoZoomer Digital Pathology System and immunoreactivity was qualitatively assessed using a 0-3+ scale, where 3+ represents the most intense staining. Electronic images of follicles at different stages of folliculogenesis were assessed by two independent observers who were blinded to the morphology of the source ovary. Each individual ovary contributed a minimum of 1 follicle per size class and a minimum of 10 follicles per size class were analysed. AR immunoreactivity was present in granulosa cells at all stages of folliculogenesis, in thecal cells of large antral follicles, and in the ovarian stroma. Staining intensity for AR did not differ between normal and polycystic ovaries. KITL expression, summarised in Table 1, was found to be significantly elevated in the oocytes of primordial and primary follicles and in the granulosa cells of follicles at all stages of folliculogenesis. These results show that AR expression is normal in PCO but expression of an AR-regulated gene is increased, potentially due to an excess of androgen hormone that is characteristic of women with PCO. Based on the roles of KITL established by murine studies, increased expression of KITL could explain many of the features of PCO including follicle excess, hyperthecosis and abnormal androgen secretion.

Identification of novel and known ovary‐specific genes including ZP2, in a marsupial, the stripe‐faced dunnart

During the early stages of oogenesis, oocyte-specific factors, synthesized by and stored within the oocyte, play critical roles during oogenesis, folliculogenesis, fertilization and early embryonic development in the mouse. The identification of marsupial maternal factors, expressed specifically in the ovary or oocyte, may provide an insight into the conserved evolutionary mechanisms that drive mammalian oocyte development to cleavage stages. In this study, 10 clones including dunnart ZP2 and c-mos, isolated by cDNA representational difference analysis, were validated by RT-PCR for ovary-specific expression. This novel combination of techniques to isolate ovary-specific genes has identified three novel genes with ovary-specific expression. Both dunnart ZP2 and c-mos exhibited ovary-specific expression, making this study the first isolation of c-mos in a marsupial species. Dunnart ZP2 expression was examined in detail by in situ hybridization and results indicate oocyte-specific expression of dunnart ZP2 in the cytoplasm of oocytes of primordial, primary and secondary follicles with expression being highest in oocytes of primary follicles. ZP2 was not expressed in granulosa cells of any follicles.