TAF4b promotes mouse primordial follicle assembly and oocyte survival

Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development.     

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.     

Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.     

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.     

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-alpha subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture (P<0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day (P<0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture (P<0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.     

Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Epiregulin-dependent amphiregulin expression and ERBB2 signaling are involved in luteinizing hormone-induced paracrine signaling pathways in mouse ovary

Sustained EGF receptor (EGFR) phosphorylation by de novo synthesis of EGFR ligands plays an essential role in mediating luteinizing hormone (LH)-induced ovulation process in the preovulatory follicles (POFs). In the present study, the effect of epiregulin (EREG) on oocyte maturation and ovulation was investigated using Ereg knockout (Ereg^−/−) mice congenic on a C57BL/6 background. Rate of spontaneous oocyte meiotic resumption of denuded oocytes (DOs) or cumulus cell-oocyte complexes (COCs) in vitro is similar between wild-type and Ereg^−/− mice. However, gonadotropin-induced meiotic resumption in vivo is attenuated, and the number of COCs with expanded cumulus matrix and superovulated eggs dramatically decrease in Ereg^−/− mice. Nonetheless, the number of eggs ovulated during normal estrus cycles and litter sizes in Ereg^−/− mice are comparable to those of wild-type littermates. In contrast to other EGFR ligands, induction of amphiregulin (Areg) mRNA is severely reduced in ovaries collected from Ereg^−/− mice either after human chorionic gonadotropin (hCG) treatment in immature mice or LH surge in adults. Gonadotropin-induced EGFR and ERBB2 phosphorylation in ovaries is attenuated in immature Ereg^−/− mice, and MAPK3/1 phosphorylation and prostaglandin synthase 2 (PTGS2) protein levels are reduced. This attenuation, however, is no longer detectable in adult Ereg^−/− mice after LH surge. This study implicates that EREG mediates signals downstream of Areg mRNA expression and that EGFR-ERBB2 signals contributes to regulation of ovulation process.     

Heteromeric complex formation of ASK2 and ASK1 regulates stress-induced signaling

Apoptosis signal-regulating kinase 2 (ASK2) is an interaction partner of the highly related ASK1. Here, we describe a regulatory function of ASK2 in stress signaling-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Increased cleavage of caspase-3 and PARP was demonstrated by overexpression as well as knockdown of ASK2 after stress-induction by serum-starvation. We show that ectopically expressed ASK2 homo-oligomerized while endogenous ASK2 and ASK1 formed hetero-oligomers, which decreased upon serum-starvation. Co-expression of ASK2 and ASK1 stabilized these two proteins and reduced starvation-induced caspase-3 activation and degradation of PARP. Analysis of the intracellular localization of ASK2 exhibited a similar localization compared with ASK1 in the nucleus, cytoplasm, and in mitochondria. We propose that ASK2 regulates stress-induced caspase-3 and PARP cleavage in a dose-dependent manner by heteromeric complex formation with ASK1.     

Enhanced BMP signaling results in supernumerary tooth formation in USAG-1 deficient mouse

Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We previously reported that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. We confirmed that BMPs were expressed in both the epithelium and mesenchyme of the rudimentary incisor at E14 and E15. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Wnt signaling as demonstrated by the nuclear localization of β-catenin was also up-regulated. Inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signaling results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1 deficient mouse model.     

新生小鼠卵巢移植雄鼠肾囊下卵泡的生长发育

将1日龄小鼠卵巢移植入成年雄鼠肾囊下,分别于移植后18d、36d回收移植卵巢进行形态学、组织学观察,以评价卵巢移植体在成年雄性受体小鼠体内生长及卵泡发育潜能。结果表明:移植体生长增大,有各级生长卵泡发育;18日龄移植体平均直径为1881.1μm±204.7μm,与1日龄卵巢相比差异极显著(P<0.01),卵泡发育到有腔卵泡阶段;36日龄移植体平均直径达2575.3μm±466.4μm,显著大于18日龄移植体(P<0.01),有成熟卵泡出现,未观察到黄体;从移植体分离到GV期卵母细胞和卵丘卵母细胞复合体。研究表明1日龄小鼠卵巢移植体在雄性受体生理环境中具有正常生长发育和形成成熟卵泡的潜能。     

c-src在大鼠卵巢中的表达及其在原始卵泡生长启动中的作用初探

目的：研究原癌基因c-src在大鼠卵巢的表达,及其在原始卵泡启动过程中的作用。方法：取2日龄SD雌性大鼠卵巢,在Waymouth培养体系中培养0．4、8d后,首先采用RT-PCR方法证实大鼠卵巢中有c-src的表达,再体外合成其RNA小干扰片段（small interference RNA,siRNA）转染培养中的卵巢组织进行RNA干扰,用HE染色及RT-PCR筛选最佳干扰片断并用慢病毒包装后检测干扰效果。结果：随着培养天数的增加,原始卵泡在卵泡总数中所占比例逐渐减少;c—src mRNA在原始卵泡中有表达,经筛选用最佳干扰片断siRNA1慢病毒包装进行RNA干扰,发现干扰后,与空白组、空白载体组相比,最佳干扰组c—src mRNA含量明显下降,原始卵泡在卵泡总数中所占比例相对更多,原始卵泡发育受到抑制。结论：c-src在原始卵泡中有表达,并在一定程度上促进了原始卵泡的发育。     

Estradiol regulates N-cadherin mRNA levels in the mouse ovary

N-cadherin (N-cad) is a calcium-dependent cell adhesion molecule which is present in the granulosa cells of the mouse ovarian follicle. This cell adhesion molecule has been implicated as a key modulator of follicular development. The regulators of N-cad mRNA levels in the ovary have not been identified. We have examined the ability of steroids to influence ovarian N-cad mRNA levels in vivo. Immature mice were injected with either progesterone, testosterone, 17β-estradiol, or 17α-estradiol. Only 17β-estradiol caused a rapid and significant increase in the ovarian N-cad mRNA levels. We speculate that this steroid is a major regulator of N-cad-mediated granulosa cell interactions in vivo. © 1995 Wiley-Liss, Inc.     

Bucky ball functions in Balbiani body assembly and animal-vegetal polarity in the oocyte and follicle cell layer in zebrafish

The Balbiani body is an evolutionarily conserved asymmetric aggregate of organelles that is present in early oocytes of all animals examined, including humans. Although first identified more than 150 years ago, genes acting in the assembly of the Balbiani body have not been identified in a vertebrate. Here we show that the bucky ball gene in the zebrafish is required to assemble this universal aggregate of organelles. In the absence of bucky ball the Balbiani body fails to form, and vegetal mRNAs are not localized in oocytes. In contrast, animal pole localized oocyte markers are expanded into vegetal regions in bucky ball mutants, but patterning within the expanded animal pole remains intact. Interestingly, in bucky ball mutants an excessive number of cells within the somatic follicle cell layer surrounding the oocyte develop as micropylar cells, an animal pole specific cell fate. The single micropyle permits sperm to fertilize the egg in zebrafish. In bucky ball mutants, excess micropyles cause polyspermy. Thus bucky ball provides the first genetic access to Balbiani body formation in a vertebrate. We demonstrate that bucky ball functions during early oogenesis to regulate polarity of the oocyte, future egg and embryo. Finally, the expansion of animal identity in oocytes and somatic follicle cells suggests that somatic cell fate and oocyte polarity are interdependent.     

Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary

The number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-1) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-1-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination.     

An ultrastructural study of relationships between the ovarian haemal system,follicle cells,and primary oocytes in the sea star,Asterias rubens

Summary The genital haemal sinus, present throughout the gonad wall of sea stars, is supposed to be the site of ultimate accumulation of nutrients for the germinal epithelium. Early vitellogenic pear-shaped oocytes are attached to this sinus by stalk-like processes. The ultrastructure of this association and of the oocyte-follicle cell complex is described with emphasis on mechanisms involved in oocyte nutrition.The genital haemal sinus, and sometimes portions of the surrounding genital coelomic sinus, contain a fine granular ground substance and amoeboid cells. Material similar to the haemal ground substance also fills vacuities in the inner basal laminae of the haemal sinus and intervenes between this layer and adjacent germinal and follicle cells in the ovarian lumen.Vitellogenesis is first detectable as numerous vacuoles accumulate within the oocyte-stalk near the haemal sinus; they contain flocculent material and often fuse with adjacent lysosome-like vacuoles. As vitellogenesis proceeds, oocytes develop complex and tenuous connections with the haemal sinus. These consist of a network of pseudopodia that interdigitate with thin sheet-like extensions of follicle cells. These cells are attached to the oolemma by microfilamentous processes and contain regularly arranged concentrations of glycogen granules and well developed rough endoplasmic reticulum.It is concluded, (1) that follicle cells provide each oocyte with a compartmentalized microenvironment within the ovarian lumen, (2) that such compartments are intimately associated with the nutrient laden haemal sinus, and (3) that nutritive and vitellogenic substances, derived extragonadally and stored temporarily in the ovarian wall, can pass through the oocyte-stalk.     

FGF22 signaling regulates synapse formation during post‐injury remodeling of the spinal cord

The remodeling of axonal circuits after injury requires the formation of new synaptic contacts to enable functional recovery. Which molecular signals initiate such axonal and synaptic reorganisation in the adult central nervous system is currently unknown. Here, we identify FGF22 as a key regulator of circuit remodeling in the injured spinal cord. We show that FGF22 is produced by spinal relay neurons, while its main receptors FGFR1 and FGFR2 are expressed by cortical projection neurons. FGF22 deficiency or the targeted deletion of FGFR1 and FGFR2 in the hindlimb motor cortex limits the formation of new synapses between corticospinal collaterals and relay neurons, delays their molecular maturation, and impedes functional recovery in a mouse model of spinal cord injury. These results establish FGF22 as a synaptogenic mediator in the adult nervous system and a crucial regulator of synapse formation and maturation during post‐injury remodeling in the spinal cord.