Follicular Fluid Of Preovulatory Follicles Research Articles

Management of ovarian functions by melatonin

Extensive research has unraveled a niche for melatonin that is of great significance for the female reproductive physiology. The potency of melatonin as an antioxidant, anti-inflammatory, and anti-apoptotic agent is being utilized to benefit female reproductive anomalies. Melatonin receptors have been localized in the Supra Chaismatic Nucleus (SCN), pars tuberalis (PT), and the gonads suggesting the regulation of reproduction by melatonin not only at a higher level but also on the gonads through complex interrelated mechanisms. Melatonin secreted by the pineal gland acts on the hypothalamus to regulate gonadotropin-releasing hormone and subsequently gonadotropin (FSH/LH) release from the PT. However, the de novo synthesis of this indoleamine reported in the gonads gave rise to the idea of a more localized action. The mammalian ovary has all the molecular machinery required for the biosynthesis of melatonin and interestingly concentration of melatonin in the follicular fluid of pre-ovulatory follicles is much higher than circulatory melatonin even in humans. This locally produced melatonin has been shown to modulate various pathways governing ovarian steroidogenesis. Further, melatonin and its receptors play a significant role in antioxidant defense mechanism of ovary for follicular growth and maturation. Exposure to stress strongly influences hypothalamic-pituitary-adrenal axis and elevated glucocorticoid levels suppress various ovarian functions including implantation thereby pregnancy. Melatonin acts antagonistically with glucocorticoids, making it crucial for the management of the female reproductive functions/dysfunctions. Usage of melatonin during in vitro fertilization (IVF) procedures has been found to improve oocyte quality, survival, and fecundity. Therefore, in future, melatonin can be implicated as preferable therapeutic especially in IVF and assisted reproductive techniques.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Effects of follicular fluid of preovulatory follicles of repeat breeder dairy cows with subclinical endometritis on oocyte developmental competence

The aims of the present study were to determine the concentrations of lipopolysaccharide (LPS), hormonal progesterone, estradiol-17β, insulin growth factor (IGF-1) and magnesium in the serum and the preovulatory follicle follicular fluid (FF) in repeat breeder (RB) cows without (nSCE) or with subclinical endometritis (SCE), and further to examine the effects of this FF on developmental competence of cattle oocytes. In Experiment 1, 13 of 23 clinically healthy Holstein RB cows were identified (uterine PMNs) to have SCE. The cows were estrous synchronized, and 6–12 h after detection of standing estrus, FF and blood of the preovulatory follicles were collected. The mean (±SD) LPS (862.3 ± 148.1 compared with 1063.4 ± 262.8 EU/ml, P = 0.04) and estradiol-17β (188.9 ± 15.8 compared with 162.0 ± 31.5 ng/ml, P = 0.02) concentrations of FF was different between nSCE and SCE cows. In Experiment 2, FF of RB cows with relatively lesser (nSCE, n = 4) and greater (SCE, n = 4) percentages of uterine PMNs was separately added to the oocyte maturation medium for in vitro embryo production. Addition of FF from SCE cows to the oocyte maturation medium resulted in a lesser rate of development to the blastocyst stage than that of the nSCE cows (21.9 ± 1.8 compared with 27.8 ± 2.5%, P < 0.05). Results of the present study indicate greater FF LPS concentration may result in a lesser quality microenvironment milieu for the final stages of oocyte maturation in RB dairy cows with subclinical endometritis. In addition, supplementation of oocyte maturation medium with FF of preovulatory follicles from RB cows with subclinical endometritis resulted in a lesser potential of in vitro oocyte developmental competence.

Involvement of Matrix Metalloproteinases and their Inhibitors in Bovine Cystic Ovarian Disease

The most important regulators of tissue remodelling during ovarian follicular growth, development, ovulation and atresia are gonadotropins, steroid hormones, growth factors and different proteolytic enzymes. Matrix metalloproteinases (MMPs) such as collagenase or gelatinase (i.e. MMP-1, -8, -2 and -9) and associated tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4) control connective tissue remodelling during follicular rupture. In this study, we hypothesized that an imbalance in the MMP-TIMP system may be an intra-ovarian component that contributes to the pathogenesis of cystic ovarian disease (COD) in cows. Taking into account that the control of MMP activity by TIMPs could determine their effects in both physiological and pathological conditions, MMP and TIMP mRNA and protein expression was examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively, in ovaries from control cows and cows with COD. Expression of mRNA encoding MMP-2, TIMP-1 and TIMP-2 was lower in follicular cysts than in control pre-ovulatory follicles, while the results by IHC showed this imbalance only for TIMP-2 protein expression. Additional analysis by zymography to evaluate the gelatinase activity of MMP-2 and MMP-9 demonstrated higher MMP-2 activity in follicular fluid (FF) of cysts than in FF of pre-ovulatory follicles. On the other hand, MMP-9 activity was increased in follicular cysts and absent in the FF of pre-ovulatory follicles. These findings suggest that the altered mRNA expression and activities of the MMP-TIMP system may be related to the failure in ovulation and follicular development observed in COD.

63 EQUINE FOLLICLES MODULATE CORTISOL LEVELS AND CAPABILITY OF OOCYTES TO ADAPT TO STRESS SITUATIONS

Glucocorticoids are mediators of the systemic stress response. Acute or chronic stress characterised by high cortisol concentrations in the periphery impairs reproductive function in a variety of species and therefore may affect fertility. The ovary has been shown to be a target tissue for glucocorticoids in many species, including the mare. This study hypothesised that the equine ovary possesses internal mechanisms to modulate cortisol activity and that supraphysiologic levels of glucocorticoids do not affect oocyte IVM rates. Light horse mares (n = 9) were used in this study. Growing follicles from an induced follicular wave were divided into the following groups: G1: 5–9 mm, G2: 10–14 mm, G3: 15–19 mm, G4: 20–24 mm, and G5: ≥25 mm. Follicular fluid (FF) and compact cumulus‐oocyte complexes (COCs) were obtained by ultrasound-guided transvaginal aspiration. Blood samples were collected at the beginning and the end of every aspiration session. Cortisol (DE1887, Demeditec, Kiel-Wellsee, Germany), progesterone (ADI-901–011, Enzo Life Sciences, Farmingdale, NY, USA), and corticosteroid binding globulin (CBG, MBS047353, MyBioSource, San Diego, CA, USA) concentrations were determined by ELISA. COCs (n = 80) were randomly distributed to either the control group (DMEM-F12+ medium) or the following hydrocortisone treatment groups: 0.1 µg mL–1, 1 µg mL–1, 5 µg mL–1, 10 µg mL–1. Maturation rate was assessed 30 h after incubation. Statistical analysis was performed with the SPSS Statistics 22 software. Data were analysed using one-way ANOVA, Pearson correlation, and chi-squared test. Cortisol (115.4 ± 13.3 ng mL–1) and progesterone (22.1 ± 3.1 ng mL–1) FF concentrations were higher (P &lt; 0.05) in G5 follicles than in all other groups, and were positively correlated (r = 0.8; P &lt; 0.001). Plasma concentrations of cortisol (118.6 ± 7.8 v. 120.3 ± 12.2 ng mL–1), progesterone (2.4 ± 0.5 v. 2.5 ± 0.4 ng mL–1), and CBG (11.1 ± 5.1 v. 9.9 ± 3.2 µg mL–1) did not differ before and after follicle aspiration. However, plasma CBG and progesterone were negatively correlated (r = –0.56; P &lt; 0.01). Maturation rates did not differ among groups, regardless of the hydrocortisone concentration added to the culture medium. Our results demonstrated higher cortisol concentrations in preovulatory follicles in vivo, suggesting its importance for oocyte maturation. The greater unbound cortisol available in the FF of preovulatory follicles can be indicative of the displacement of cortisol from CBG in favour of progesterone. Furthermore, equine oocytes were capable of surviving cortisol concentrations 100 times higher than those physiologically present in preovulatory follicles. This finding suggests the ability of equine oocytes to modulate cortisol levels and adapt to stress situations.

Short communication: Concentrations of the mammalian lignan enterolactone in preovulatory follicles and the correlation with intrafollicular estradiol in dairy cows fed extruded flaxseed

Flaxseed is a rich source of lignans that can be metabolized to the mammalian lignan enterolactone (EL), which may elicit weak estrogenic or antiestrogenic effects. The objectives of this study were to examine the effects of feeding an extruded flaxseed supplement to dairy cows on concentrations of EL in plasma and preovulatory follicles and the association with intrafollicular estradiol (E2). Twenty-four multiparous 256-d-pregnant Israeli Holstein cows were fed either a standard diet both pre- and postpartum (control; n=12) or provided with an extruded flaxseed supplement (n=12), at 7.9 and 9.2% of dry matter, pre- and postpartum, respectively. Follicular fluid (FF) aspirations were conducted at 84±16 d in lactation as follows: 7 to 8 d following behavioral estrus, cows were injected with prostaglandin F2α and 48h later follicles >7mm were aspirated. Follicles were regarded as preovulatory when the E2-to-progesterone ratio was >1. Plasma EL concentrations were not different between treatment groups; however, concentrations of EL in FF of preovulatory follicles were 1.7 times higher in extruded flaxseed-supplemented cows than in control. Across-treatment analysis revealed a positive correlation between concentrations of EL in plasma and in FF. In addition, intrafollicular EL concentrations were positively correlated with E2 concentrations (r=0.50), and with the intrafollicular E2-to-progesterone ratio. In conclusion, supplementing dairy cows with extruded flaxseed increased EL concentrations in preovulatory follicles. Intrafollicular EL was correlated with E2 concentrations; therefore, the possible effects of EL from flaxseed on follicular steroidogenesis should be considered.

179 CORPUS LUTEUM DEVELOPMENT AFTER FOLLICLE ASPIRATION IS RELATED TO CHARACTERISTICS OF THE ASPIRATED FOLLICLE IN DAIRY COWS

The aim of this study was to investigate whether morphology or function of a corpus luteum (CL), which developed after an aspiration of a preovulatory follicle, is related to follicular characteristics such as size or steroid content. If so, CL morphology or function could be used as a retrospective quality parameter for the aspirated follicle or the follicular characteristics as a prospective parameter for the quality of the developing CL. Therefore, 44 aspiration sessions were performed using 18 cows between 26 and 121 days after parturition during the first lactation. Heat was induced in mid-dioestrus with Cloprostenol. A GnRH analogon (Depherelin) were administrated 54 h later. The dominant follicle was aspirated 21 h after administration of the GnRH analogue. The diameter of the dominant follicle at aspiration and the cross-section area of the resulting luteal tissue 14 days later were measured by ultrasound. Concentrations of progesterone (P4) and 17-β-oestradiol (E2) in blood and follicular fluid (FF) were determined by H3-RIA. A CL development occurred in 82% of aspiration sessions after aspirating the dominant follicles. The interval of time between parturition and follicle aspiration did not affect variables investigated. As expected, cross-section area of the luteal tissue was positively correlated with plasma P4 concentration on Day 14 after follicle aspiration (r = 0.54; P &lt; 0.01). The diameter of the aspirated follicle was positively correlated with the plasma P4 concentration on Day 14 after follicle aspiration (r = 0.52; P = 0.02), but the diameter was not correlated with the cross-section. Comparing the FF of follicles that originated a CL after aspiration with follicles that did not, both types had comparable P4 concentrations (578 ± 79 ng mL–1 v. 398 ± 84 ng mL–1; P = 0.2), but the former type presented higher E2 concentrations (206 ± 23 ng mL–1 v. 64 ± 29 ng mL–1, P &lt; 0.01) compared with the latter. The P4 concentrations in FF of follicles that originated a CL after aspiration were positively related to cross-section area of the CL (r = 0.48; P = 0.04), but neither P4 nor E2 concentrations in the FF of preovulatory follicles could be used as a predictor for the plasma P4 concentration at day 14. In conclusion, CL development after follicle aspiration can be used as a retrospective quality parameter of aspirated dominant follicles. Preovulatory follicle diameter as well as cross-section area of the luteal tissue could be used prospectively to identify cows with high plasma P4 levels, and this may help to identify suitable recipients for embryo transfer. This study was supported by the German Research Foundation (DFG WE 2458/7-2).

Growth differentiation factor 9 concentration in the follicular fluid of infertile women with endometriosis

This study evaluated the level of growth differentiation factor 9 (GDF-9) in the follicular fluid of infertile women with endometriosis. The presence of GDF-9 in follicular fluid of preovulatory follicle was confirmed by Western blotting method in the band of 53 kDa, and GDF-9 concentration in women with severe endometriosis was lower than in those without.

Expression of AT1 (angiotensin type 1) and AT2 (angiotensin type 2) receptors in human granulosa-lutein cells (GL): correlation with the cause of infertility

OBJECTIVE: GL cells and follicular fluid of preovulatory follicles from women undergoing IVF contain high concentrations of AngII. The same GL cells undergo different degrees of apoptosis as a function of age (younger < older) and IVF outcome (pregnant < non-pregnant) (Bencomo et al. Fert & Steril. 2006). AngII induces in vitro apoptosis of gonadotrophin-exposed human IVF GL cells (Fert & Steril, in press). The aim of this study is to determinate the expression pattern of AT1 and AT2 receptors in GL cells in patients with different infertility diagnosis. DESIGN: AT1 and AT2 expression was assessed by qRT-PCR. MATERIALS AND METHODS: GL cells from 69 IVF-ICSI cycles were analyzed. Patients were classified as: 1. “No ovarian factor” (tubal or male factor and egg donors, NOF; n=22); 2. Poor responders and older patients (PR; n=15); 3. Endometriosis (ENDO; n=15); 4. Polycystic ovary syndrome (PCOS; n=17). Ovarian stimulation was carried out using a long or microflare or antagonist protocol based on clinical parameters. GL cells were collected from pooled follicular fluids (FF) and purified using a 50% Percoll gradient and anti-CD45 imunobeads. AT1 and AT2 were measured by qRT-PCR as relative expression compared to beta-actin. Statistical analysis was carried out using Pearson's correlation, one way ANOVA and Student's t-test. RESULTS: Analysis of all cycles showed a higher expression of AT1 compared to AT2 (7.32 ± 0.77 vs 1.18 ± 0.33) and the existence of correlation between AT1 and AT2 expression (r=0.26; p<0.05). Separate analysis of NOF showed a positive correlation between AT1 and AT2 (r=0.52; p<0.05) and a negative correlation between AT1 and MII oocytes retrieved (r=-0.64; p<0.01). In the PCOS group there was a positive correlation between AT2 expression and patients' age (r=0.53; p<0.05). Finally, AT1 expression in PR patients was statistically significantly lower (3.87 ± 0.52) than in NOF (9.11 ± 1.72), ENDO (7.40 ± 1.63) and PCOS (7.99 ± 1.38). CONCLUSIONS: This study show a linear relationship between AT1 and AT2 expression in GL cells. Expression of AT1 is lower in poor responders, who also have increased apoptosis. The negative correlation between AT1 and MII oocytes warrants further investigation. Studies are underway to determine the relevance of these results to the outcome of IVF cycles.

Lysophosphatidic Acid Up-Regulates Expression of Interleukin-8 and -6 in Granulosa-Lutein Cells through Its Receptors and Nuclear Factor-κB Dependent Pathways: Implications for Angiogenesis of Corpus Luteum and Ovarian Hyperstimulation Syndrome

Lysophosphatidic acid (LPA) was found at significant amounts in follicular fluid of preovulatory follicle. The lysophospholipase D activity of serum from women receiving ovarian stimulation was higher than women with natural cycles. Angiogenic cytokines, including IL-6, IL-8, and vascular endothelial growth factor, increased in plasma and ascites of patients with ovarian hyperstimulation syndrome. The role of LPA in ovarian follicles is unclear. Our objective was to investigate the expression of LPA receptors and function of LPA in granulosa-lutein cells. Granulosa-lutein cells were obtained from women undergoing in vitro fertilization. We examined the expression of LPA receptors using RT-PCR. The effects of LPA on the expression of IL-6, IL-8, and vascular endothelial growth factor were examined. Signal pathways of LPA were delineated. The functions of secretory angiogenic factors were tested using human umbilical vein endothelial cells. The LPA1, LPA2, and LPA3 receptors' mRNA was identified in granulosa-lutein cells. LPA enhanced IL-8 and IL-6 expressions in a dose- and time-dependent manner. LPA functioned via LPA receptors, Gi protein, MAPK/ERK, p38, phosphatidylinositol 3-kinase/Akt, and nuclear factor-kappaB, and transactivation of epidermal growth factor receptor. LPA induced IL-8 and IL-6 through different pathways. LPA-induced IL-8 and IL-6 increased permeability of human umbilical vein endothelial cell monolayer. LPA induces IL-8 and IL-6 expressions through LPA receptors and nuclear factor-kappaB dependent pathways in granulosa-lutein cells. The LPA in preovulatory follicles may play a role in the angiogenesis of corpus luteum. Large amounts of LPA-induced IL-8 and IL-6 from multiple corpora luteae of stimulated ovaries may be one of the pathophysiological causes of ovarian hyperstimulation syndrome.

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: Relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A)

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.

Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women

Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles.

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/microliter), cumulus cells from oocytectomized complexes (1 OOX/microliter), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/microliter), or oviductal cells (1000 cells/microliter) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 microgram/ml) to microdrops of the conditioned medium. After 16-18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all states of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo.

Plasminogen Activator and Plasminogen Activator Inhibitor in Human Preovulatory Follicular Fluid*

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Periovulatory Follicular Fluid Hormone Levels in Spontaneous Human Cycles*

We measured follicular fluid hormone levels in 48 normally cycling infertile women who underwent follicle puncture and oocyte retrieval during diagnostic laparoscopy at time-bracketed intervals after an endogenous LH surge. Follicular fluid LH, FSH, PRL, estrone (E1), estradiol (E2), progesterone (P), androstenedione (A), and testosterone (T) concentrations and P/E2 and A/E2 ratios were determined. Oocytes were classified as germinal vesicle (gv), metaphase I (mI), metaphase II (mII), or degenerating (dg). Follicular fluid (ff) hormone levels then were correlated with the stage of oocyte maturation. There were no differences in ff E1 or E2 levels at any stage of oocyte maturation, except that the mean ff E2 concentration was significantly (P less than 0.05) lower in ff containing dg oocytes [2,474 +/- 1,435 (+/- SE) nmol/L] than in those containing the other oocyte stages. The mean P levels were significantly (P less than 0.0001) higher in ff containing mI (48,781 +/- 10,240 nmol/L) and mII (41,801 +/- 11,098 nmol/L) oocytes than in ff containing gv oocytes (1371 +/- 696 nmol/L). The mean A level was highest (P less than 0.01) in dg-associated ff. Similarly, T was highest (P less than 0.05) in ff containing dg (52 +/- 14 nmol/L) oocytes than in ff containing mI (10.7 +/- 10.1 nmol/L) or mII (10.1 +/- 4 nmol/L) oocytes, and it was also elevated (P less than 0.05) in gv ff (72 +/- 33 nmol/L) compared to mII ff. The above differences also were reflected in the P/E2 ratio, which was significantly higher (P less than 0.05) in mI and mII ff, as well as in the A/E2 ratio, which was higher (P less than 0.05) in ff containing mI and mII oocytes compared to ff containing gv or dg oocytes. These data define the evolving changes in the microenvironment of the follicular fluid of preovulatory follicles of normally cycling women. They also provide reference points for analysis of ff obtained from women during stimulated cycles intended for in vitro fertilization.

CAPACITATION OF HAMSTER SPERMATOZOA WITH BLOOD SERA

Yanagimachi & Chang (1964) were the first to report in vitro fertilization in the golden hamster using both epididymal and uterine spermatozoa. These studies were later extended by Barros & Austin (1967), who showed that follicular eggs could be readily fertilized in vitro with epididymal spermatozoa. They also showed that the follicular fluid of pre-ovulatory follicles could induce the acrosomal reaction at a higher rate than tubal fluid. Later, Barros (1968a) showed that a 4-hr incubation of epididymal spermatozoa with tubal fluid containing eggs was enough for the complete capacitation of hamster spermatozoa, suggesting that, in the hamster, the process of capacitation takes about this length of time. Capacitation, as judged by the occurrence of the acrosomal reaction and by the incidence of eggs fertilized in vitro, was not species-specific, since both tubal fluid containing mouse and rat eggs `in cumulus' (Barros, 1968b) and follicular fluid from the same species (Yanagimachi, 1969b) were found to capacitate hamster spermatozoa in vitro. Moreover, Yanagimachi (1969a) studied several fractions of bovine follicular fluid for the induction of capacitation and the acrosomal reaction of hamster spermatozoa, and found that there are at least two fractions in this fluid which are important in the process of capacitation; one fraction is dialysable and heat stable at 90\s=deg\C and has sperm-activating properties and the other is non-dialysable and heat labile at 90\s=deg\C and is responsible for the acrosomal reaction. Caravaglios 8c Cilotti (1957) showed that bovine follicular fluid and blood serum have similar electrophoretic patterns when subjected to paper electrophoresis. Using zone electrophoresis and immuno-electrophoresis, Marcus & Saravis (1965) found the same five protein components in the tubai fluid and in the blood serum of the rhesus monkey, in approximately the same percentage ranges. Albers & Castro (1961) demonstrated the presence ofat least five protein components in rat uterine fluid. One of them has a /?-globulin mobility and is specific for rat uterine fluid. Later studies made by Kunitake, Nakamura, Wells & Moyer ( 1965) revealed nine protein components in rat uterine fluid. They found at least four proteins which are common to rat serum and five protein components which are native to uterine fluid. The resemblances between blood serum and the female genital tract secre¬ tions prompted us to investigate whether blood serum could also induce sperm capacitation and the acrosomal reaction.