甲状腺激素受体在扬子鳄卵巢中的免疫组织化学定位和不同繁殖时期的mRNA表达水平变化检测

目的研究扬子鳄甲状腺激素受体(thyroid hormone receptor,TR)基因的系统进化关系和在卵巢组织中的定位,分析成年扬子鳄在不同繁殖活动期卵巢组织中甲状腺激素受体的动态变化规律。方法通过Clustal W软件对扬子鳄和其它脊椎动物TRs氨基酸序列进行序列比对,使用MEGA 6.0软件构建NJ系统发育树;应用q RT-PCR方法检测TRα和TRβm RNA在不同繁殖时期卵巢组织中的表达水平差异;应用免疫组织化学法对扬子鳄卵巢卵泡中TR进行组织定位。结果系统发育分析显示扬子鳄与鸟类亲缘关系较近;扬子鳄卵巢中TR的m RNA表达水平在繁殖期最高,在繁殖前期和繁殖后期相对较低;免疫组织化学染色显示,TR阳性反应主要位于卵泡的颗粒层细胞、卵泡膜、卵母细胞质和卵母细胞核中,其中颗粒层细胞阳性反应最明显。结论 TR基因的进化时间较早,因此序列保守性较高;TRα和TRβ对扬子鳄卵泡发育成熟具有重要作用;颗粒层细胞是TRs的功能作用主要位点。     

性类固醇激素及其受体在文昌鱼性腺和神经系统中的分布

首次用雌二醇,睾酮及孕酮受体的多克隆抗体在文昌鱼性腺,脑泡和神经管中进行免疫细胞化学定位研究,结果表明,不同发育时期的卵原细胞和卵母细胞中都存在雌激素,雄激素和孕激素受体,在小生长期,3种受体通常定位在卵原细胞和早期初级卵母细胞的胞质或核膜,在大生长期和成熟期,则定位在胞质和核质,在雄性,3种类固醇激素受体分布在不同发育时期的精原细胞,精母细胞和精子细胞（雌激素受体例外）中,受体阳性物定位在胞质和核,另外,用免疫细胞化学方法和性类固醇激素抗体对文昌鱼不同发育时期的性腺和神经系统进行研究,结果揭示,免疫阳性物存在于不同发育时期的卵巢和精巢中,在卵巢发育是上期,卵原细胞和卵母细胞的胸质和核仁膜对紫二醇,睾酮和孕酮抗体显示强或中等强度免疫阳性反应,在大生长期和成熟期,卵母细胞胞质和核仁膜对睾酮和孕酮抗体的免疫阳性反应明显减弱,而对雌二醇则显著增强,在雄性,随着精原细胞和精母细胞对孕酮抗体发生强的免疫阳性反应,在成熟期则对雌二醇抗体发生免疫阴性反应,精子始终为免疫阴性,本文研究首次发现,文昌鱼脑泡和神经管中存在哺乳动物神经旮体激素样免疫阳性神经细胞及其纤维,免疫阳性物分布在神经细胞胸质,核显阴性反应,这些结果为证明文昌鱼性类固醇激素参与调节性腺发育成熟和生殖内分泌调控轴的功能成熟提供重要的形态学新证据。     

干细胞因子在卵泡发育过程中的作用

干细胞因子(stemcellfactor,SCF)是酪氨酸激酶受体的配体。哺乳动物卵巢组织能表达SCF,它不仅能促进和诱导卵母细胞的发育,并能调控卵泡细胞间的相互作用及激素的生成,而且是卵泡发育过程中重要的旁分泌因子,可能激活蛋白激酶C(PKC)和有丝分裂原激活蛋白激酶的激酶(MEK)及PI3激酶途径信号分子等信号途径,对卵泡发育起调节作用。     

VEGF在人胎卵巢发育过程中的表达研究

目的研究血管内皮生长因子（VEGF）在人胚胎卵巢组织发生过程中的表达特征,探讨其在卵巢发生中的作用。方法采用HE染色和SP免疫组织化法学法检测VEGF在不同胎龄卵巢组织中的表达变化。结果VEGF在胎儿卵巢初级卵母细胞、卵泡细胞、部分基质细胞呈阳性表达,在卵母细胞的染色程度均强于卵泡细胞和基质细胞,基质小血管内皮也有阳性表达。其在卵母细胞中以胎24w阳性细胞多且表达量强,此后呈逐渐下降趋势。结论胎儿卵巢存在局部调节因子,VEGF表达于人胎卵巢中,以自分泌或旁分泌方式参与卵母细胞生长,在卵巢发生、发育过程中起着一定的作用。     

短额负蝗卵子发生过程中糖复合物的动态分布

以生物素标记的凝集素(UEA-I、SBA、PNA)为探针,利用凝集素组织化学方法对短额负蝗(Atracto-morphasinensis)卵子发生过程中滤泡细胞和卵母细胞内糖复合物的分布进行了定位研究。结果表明,在卵子发生的各期滤泡细胞和卵母细胞中没有UEA-I受体的表达,SBA和PNA受体以不同的分布模式呈阶段性表达。两者首次出现于卵母细胞生长期,随后PNA受体消失,SBA受体大量表达;在卵黄形成期前期SBA受体和重新出现的PNA受体表达于卵黄颗粒形成部位,卵黄形成期后期两者均为阴性表达;成熟卵子中两种受体又以不同程度重新出现于卵黄膜。两种受体在滤泡细胞内均大量表达。提示,N-乙酰半乳糖胺和半乳糖-β-(1,3)半乳糖胺复合物的修饰和变化与卵母细胞的发育、卵黄物质的形成及滤泡细胞的增殖分化密切相关,卵黄膜上的糖复合物可能与精卵识别有关。     

短额负蝗卵子发生过程中糖复合物的动态分布

以生物素标记的凝集素(UEA-I、SBA、PNA)为探针，利用凝集素组织化学方法对短额负蝗(Atractomorpha sinensis)卵子发生过程中滤泡细胞和卵母细胞内糖复合物的分布进行了定位研究。结果表明，在卵子发生的各期滤泡细胞和卵母细胞中没有UEA-I受体的表达，SBA和PNA受体以不同的分布模式呈阶段性表达。两者首次出现于卵母细胞生长期， 随后PNA受体消失，SBA受体大量表达；在卵黄形成期前期SBA受体和重新出现的PNA受体表达于卵黄颗粒形成部位，卵黄形成期后期两者均为阴性表达；成熟卵子中两种受体又以不同程度重新出现于卵黄膜。两种受体在滤泡细胞内均大量表达 提示，N-乙酰半乳糖胺和半乳糖-β-(1，3)半乳糖胺复合物的修饰和变化与卵母细胞的发育、卵黄物质的形成及滤泡细胞的增殖分化密切相关，卵黄膜上的糖复合物可能与精卵识别有关。     

卵母细胞在哺乳动物卵巢排卵过程中的决定性作用

哺乳动物卵巢排卵是一个复杂的调控过程。卵泡成熟破裂后,卵母细胞从卵巢中排出。卵泡细胞感受排卵刺激,并诱导卵母细胞减数分裂的恢复及其随后的释放。卵母细胞及其周围颗粒细胞的旁分泌在对此起关键性作用,其中卵母细胞对其释放具有决定性作用。作者先前已经阐述过颗粒细胞在哺乳动物卵巢排卵过程中的调控作用,该文将从卵母细胞的发育及其调控角度重点阐明其在排卵过程中的决定作用,旨在进一步理解哺乳动物卵巢的排卵过程,同时为不孕不育等卵巢疾病的治疗提供重要的研究方向和理论基础。     

小鼠卵巢促性腺激素受体的免疫组化定位

目的探明小鼠两种促性腺激素受体(FSHr、LHr)在卵巢的位置分布,揭示促性腺激素(GTH)调节卵巢机制及与卵泡发育分化的关系。方法运用免疫组化ABC法对小鼠卵巢FSHr、LHr分别进行定位染色,结合图像分析系统处理分析阳性切片。结果①FSHr阳性物质主要见于GC、TC、卵母细胞及间质细胞。随卵泡的发育,FSHr、LHr阳性细胞数量呈增长趋势,卵泡早期与中期之间阳性细胞数量差异显著,平均吸光度变化差异不显著。②LHr阳性物质主要见于卵泡TC、间质细胞、GC、卵母细胞,阳性物质着色以卵泡中、晚期较强,阳性细胞数量以卵泡中期与晚期之间差异显著。平均吸光度变化差异不显著。结论卵泡颗粒细胞、膜细胞上受体是接受促性腺激素的主要调节部位,受体数量与卵泡大小和发育程度有一定的正相关。     

c-fos-like蛋白在尖唇散白蚁繁殖蚁和工蚁性腺中的表达

为了探讨c-fos原癌基因在白蚁生殖品级和非生殖品级性腺发育中的作用,揭示白蚁不同品级性腺发育的调节机理,本研究运用免疫细胞化学定位方法对尖唇散白蚁Reticulitermes aculabialis繁殖蚁和工蚁精、卵发生过程中的c-fos蛋白表达进行了研究。结果显示：雌性繁殖蚁在末龄若虫期的卵子发生过程中有c-fos-like表达, c-fos-like免疫阳性物质定位于生长期的卵母细胞核和滤泡细胞核中;而繁殖蚁成虫的卵子发生过程中没有c-fos-like免疫阳性反应。雄性繁殖蚁在末龄若虫期时精子发生过程中没有c-fos-like表达, 而发育到成虫期有c-fos-like免疫阳性反应,阳性物质定位于精原细胞的细胞核中。工蚁精、 卵发生过程中均没有c-fos-like的表达。结果提示：c-fos在调节繁殖蚁精子和卵子发生方面有重要作用,c-fos可能通过调节精原细胞增殖参与精子的发生;在卵巢中可以直接作用于生长期的卵母细胞和滤泡细胞来调节卵子的发生。在工蚁性腺中c-fos表达缺失可能导致卵母细胞和滤泡细胞无法正常发育,精原细胞停止增殖而使精子发生处于相对抑制状态。工蚁性腺退化不育可能与c-fos没有正常表达有关。     

南方鲶卵巢滤泡细胞和卵膜生成的组织学研究

南方鲶的卵巢滤泡细胞源于卵巢基质细胞,从发生到退化分为零散卵泡膜细胞期、单层扁平泡膜细胞期、多层扁平卵泡膜细胞期、立方形颗粒细胞期柱状颗粒细胞期、颗粒细胞分泌期和颗粒细胞退化期。精孔细胞中发育中滤泡细胞分化形成。初级卵精源于卵母细胞,次级卵膜由晚期滤泡细胞分泌形成。本文还对滤泡细胞和卵膜的作用进行了阐述。     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.     

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early-spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β-estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β-estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley-Liss, Inc.     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.     

In-vitro Production of Meiosis Inducing Substance (MIS) by Isolated Amphibian (Rana pipiens) Follicle Cells*

Previous studies indicated that pituitary hormone induced oocyte maturation in preovulatory amphibian ovarian follicles is mediated by somatic elements of the follicle. In this study procedures were developed for isolating and culturing follicle cells and their ability to produce meiosis inducing substance (MIS) was assessed. Defolliculated oocytes surrounded by a single layer of follicle cells but not denuded oocytes matured in response to frog pituitary hormone (FPH) stimulation. Cultured follicle cells secreted MIS following stimulation with FPH. The amount of MIS activity produced was related to the number of follicle cells cultured and the dose of FPH utilized. Radioimmunoassay (RIA) analysis of medium from follicle cell cultures demonstrated that FPH stimulated steroid (progesterone) secretion from these cells. Addition of cAMP to follicle cell cultures enhanced FPH stimulated steroid production. The results indicate that follicle cells retain FPH responsiveness when uncoupled from the immature oocyte and exhibit both MIS and steroid secretory functions.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.