Preovulatory Period Research Articles

A Doppler ultrasonographic study of cyclic changes of ovarian perfusion in the Beagle bitch

Changes in intraovarian arterial blood flow were monitored by means of colour-coded and pulsed Doppler ultrasonography in Beagle bitches during the normal oestrous cycle (n = 11) and pregnancy (n = 3), and at PGF(2alpha)-induced luteolysis (n = 4). The ultrasonographic findings were related to the reproductive stage of the bitch, as determined by vaginoscopical and cytological criteria, and by the concentrations of oestradiol, LH and progesterone in peripheral blood plasma. Colour-coded Doppler ultrasonography was used to visualize and estimate intraovarian vascularization, and pulsed Doppler ultrasonography was used to measure the arterial blood flow. The systolic and diastolic peak velocities, the end-diastolic velocity, and the pulsatility index and resistance index were calculated for quantitative analysis of the Doppler waveforms. Intraovarian perfusion increased gradually during pro-oestrus. A marked enhancement of intraovarian colouring and blood flow velocities, and a decline in the indices for pulsatility and resistance were observed in the preovulatory period. Maximum perfusion was observed at ovulation and during the early luteal phase. Significant differences (P < 0.05) were detected for the values of all calculated Doppler parameters 2 days before and 2 days after ovulation. Intraovarian blood flow decreased gradually in accordance with luteal regression. Treatment with PGF(2alpha) caused a distinct decline in luteal activity and a concomitant reduction in intraovarian perfusion. The values of blood flow parameters found during the luteal phase of pregnant bitches were comparable to those of the normocyclic bitches. Doppler ultrasonography of the intraovarian arteries in bitches provides complementary information about cyclic changes of ovarian function.

In vivo ovarian responses to recombinant follicle-stimulating hormone (FSH) with and without recombinant luteinizing hormone (LH) in polycystic ovary syndrome (PCOS).

Objective: To contrast the effects of recombinant FSH (rFSH) with the added effects of recombinant LH (rLH) in a 1:1 ratio on ovarian secretion of 17α-hydroxyprogesterone (17OHP), androstenedione (δ4) and testosterone (T) in women with PCOS during ovulation-induction. Design: Prospective, controlled study in a University-based fertility centre. Materials/Methods: Forty-eight clomiphene-resistant women with PCOS were allocated to receive either daily injections of rFSH (n = 24) or rFSH+LH (n = 24) in a 1:1 ratio starting: 1) on day 2–3 of progestogen-induced menses (n = 8); 2) after 6 weeks of GnRH agonist treatment (nafarelin, 400 μg b.i.d; n = 8); or 3) after nafarelin treatment as in (2) plus dexamethasone (n = 8). After the initial 5-day fixed gonadotropin regimens, the dosage was adjusted according to serum E2 levels and follicle size and number on transvaginal ultrasound assessment. hCG was given [Congruent]24 hours after the last gonadotropin dose to trigger ovulation when the E2 levels were 2000 pg/ml and at least 1 leading follicle ≥15 mm (mean diameter) was seen on ultrasound examination. Between-regimen and within-subgroup interactions in serum levels of LH, 17OHP, δ4 and T were evaluated by ANOVA with specific treatment days as a repeated measures factor. Results: Pre-study hormone levels were similar for all groupings. Nafarelin significantly suppressed LH levels, which remained at the lower limit of assay sensitivity (0.5 IU/L) during stimulation with rFSH but increased significantly to >1 IU/L when rLH was added. Independent of exogenous rLH treatment, there was a small, but uniform increase in 17OHP, δ4 and T levels in the pre-ovulatory period. As expected, a greater increase occurred in subjects on rFSH + rLH. Dexamethasone further suppressed 17OHP, δ4 and T levels and unmasked a small, but significant rise in these ovarian steroids 24 hours following the first dose of rFSH + rLH, a rise that was absent with rFSH alone. Secretion of these steroids then appeared to “catch-up” after 5 days of rFSH stimulation. Conclusions: The differing secretion patterns would suggest greater reliance on compensatory paracrine mechanisms in production of these steroids when only rFSH is given following pituitary/adrenal suppression. Ideal ranges for LH levels during gonadotropin treatment remain to be explored but suppression of LH to suboptimal levels may increase the vulnerability of some of the processes involved in follicle development in the in vivo setting. Supported by: This work was supported by a research grant from the University of Alberta and Hospitals and Serono Canada Inc.

Seasonal variation in plasma lipids, lipoproteins, apolipoprotein A-I and vitellogenin in the freshwater turtle, Chrysemys picta

An analysis of plasma lipids and lipoprotein fractions was performed over the course of the annual ovarian cycle of the female turtle, Chrysemys picta. Determinations of total plasma triglycerides, cholesterol, vitellogenin and apolipoprotein A-I (apoA-I) were made. The lipid and protein composition of the lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and very high density lipoprotein (VHDL)] were also observed over the same period. Plasma triglyceride and vitellogenin levels were significantly increased in the spring preovulatory period and fall recrudescent phase. Total plasma cholesterol levels were significantly elevated only at the onset of the fall recrudescent phase and apoA-I levels were highest during the postoviposition/ovarian arrest phase. The triglyceride content of VLDL was highest in preovulatory animals and there were apparent seasonal changes in the expression of apoA-I and apoE of HDL/VHDL. We conclude that the coordinate regulation of lipids and protein contributes to seasonal ovarian growth and clearance of lipids from plasma, both of which are most likely under hormonal control.

Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation.

During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.

Alteration of gonadotrophin and steroid hormone release, and of ovarian function by a GnRH antagonist in gilts

This study examined the impact of the gonadotrophin-releasing hormone (GnRH) antagonist Antarelix™ on LH, FSH, ovarian steroid hormone secretion, follicular development and pituitary response to LHRH in cycling gilts. Oestrous cycle of 24 Landrace gilts was synchronised with Regumate ® (for 15 days) followed by 800 IU PMSG 24 h later. In experiment 1, Antarelix™ ( n=6 gilts) was injected i.v. (0.5 mg per injection) twice daily on four consecutive days from day 3 to 6 (day 0=last day of Regumate ® feeding). Control gilts ( n=6) received saline. Blood was sampled daily, and every 20 min for 6 h on days 2, 4, 6, 8 and 10. In experiment 2, gilts ( n=12) were assigned to the following treatments: Antarelix™; Antarelix™ + 50 μg LHRH on day 4; Antarelix™ + 150 μg LHRH on day 4 or control, 50 μg LHRH only on day 4. Blood samples were collected daily and every 20 min for 6 h on days 2, 4 and 6 to assess LH pulsatility. Ovarian follicular development was evaluated at slaughter. Antarelix™ suppressed ( P<0.05) serum LH concentrations. The amount of LH released on days 4–9 (experiment 1) was 8.80 versus 36.54 ng ml −1 (S.E.M.=6.54). The pattern of FSH, and the preovulatory oestradiol rise was not affected by GnRH antagonist. Suppression of LH resulted in a failure ( P<0.05) of postovulatory progesterone secretion. Exogenous LHRH (experiment 2) induced a preovulatory-like LH peak, however in Antarelix™ treated gilts the LH surge started earlier and its duration was less compared to controls ( P<0.01). Furthermore, the amount of LH released from day 4 to 5 was lower ( P<0.01) in Antarelix™, Antarelix™ + 50 and Antarelix™ + 150 treated animals compared to controls. No differences were estimated in the number of LH pulses between days and treatment. Pulsatile FSH was not affected by treatment. Mean basal LH levels were lower ( P<0.05) after antagonist treatment compared to controls. Antarelix™ blocked the preovulatory LH surge and ovulation, but the effects of Antarelix™ were reduced by exogenous LHRH treatment. The development of follicles larger than 4 mm was suppressed ( P<0.05) by antagonist treatment. In conclusion, Antarelix™ treatment during the follicular phase blocked preovulatory LH surge, while FSH and oestradiol secretion were not affected. Antarelix™ failed to alter pulsatile LH and FSH secretor or pituitary responsiveness to LHRH during the preovulatory period.

Mechanism of action of hormonal preparations used for emergency contraception: a review of the literature

This paper focuses on the research efforts undertaken to understand how emergency contraception (EC) methods act to prevent pregnancy and to identify what is known and what are the important gaps that need to be addressed. Divided into five sections the first section presents a discussion on the background of the review and a brief description of the mode of use efficacy and most common side effects of the Yuzpe regimen levonorgestrel (LNG) and mifepristone. Section 2 includes studies on the effects of postcoital steroid administration upon fertility in nonprimate animal models. Section 3 focuses on the effects of estrogens progestins or the antiprogestin mifepristone administered in the preovulatory period to macaques and the New World monkey Cebus apella. Section 4 highlights clinical studies on the effects of the Yuzpe regimen administered before and after the luteinizing hormone surge and on progesterone-regulated endometrial proteins. Finally section 5 identifies some of the most difficult areas of the literature that need to be researched.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Decreases Responsiveness of the Hypothalamus to Estradiol as a Feedback Inducer of Preovulatory Gonadotropin Secretion in the Immature Gonadotropin-Primed Rat

Sprague–Dawley rats (23-day-old) were dosed with TCDD (32 μg/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected (5 IU, sc) 24 h later to induce follicular development. Another 24 h later, half of TCDD- or corn oil-treated rats were injected (sc) with 17β-estradiol-cypionate (ECP, at 0.004 to 0.5 mg/kg). Blood and ovaries were collected on expected proestrous (preovulatory period) at 51, 54, and 58 h after eCG injection as well as in the morning after ovulation (72 h after eCG). Serum concentrations of 17β-estradiol (E), progesterone (P), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. The number of ova shed was measured at 72 h after injection of eCG by irrigating ova from oviducts. During the preovulatory period (∼58 h after eCG injection), a circulating level of 70–100 pg E/ml coincided with LH and FSH surges and later normal ovulation of 10 to 12 ova/rat was observed in controls. However, the same concentration of E was not associated with LH and FSH surges in rats treated with TCDD (32 μg/kg), resulting in reduced ovarian weight gain and reduction of ovulation by 70 to 80% (2–3 ova/rat). Blockage of the gonadotropin surge, reduced ovarian weight gain, and ovulation were all reversed completely by the lowest effective dose of ECP (0.1 mg/kg). At 72 h after eCG, serum P secretion was reduced and serum E levels were significantly increased compared to those of corn oil-treated controls. ECP alone had no effect on serum P levels at any time point, but in rats treated with TCDD and ECP, both the reduction of P (at 58 and 72 h) and the increase in E secretion (72 h) were completely reversed. Further studies confirmed that restoration by ECP of gonadotropin surges and associated ovulation could not be attained until circulating levels of E rose sufficiently high to trigger the LH and FSH surges. The new action threshold of E for inducing gonadotropin surges in rats treated with TCDD (32 μg/kg) was determined to be eight- to 10-fold higher than that in controls. Thus, it is apparent that TCDD decreased the responsiveness of the hypothalamus to E as a feedback inducer of preovulatory gonadotropin secretion.

Role of Meiosis-Activating Sterols in Rat Oocyte Maturation: Effects of Specific Inhibitors and Changes in the Expression of Lanosterol 14α-Demethylase During the Preovulatory Period1

In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P(450) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1-200 microM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.

Physiological and genetic differences between low and high index dairy cows

AbstractSelection of dairy cattle for increased milk production has decreased some indices of reproductive efficiency. For example, days open are increased by one day for every 100 kg of increased milk yield per lactation. Some of the change in days open can be explained by delayed onset of oestrous cyclicity and lower conception rate to artificial insemination in cows with greater milk production. Despite these negative associations between milk production and reproduction, reproduction in herds of high producing dairy cattle is not necessarily compromised relative to reproduction in herds of low producing dairy cattle. This is because there is a large environmental effect on dairy reproduction. High producing herds generally have better management and better oestrous detection. Therefore, high producing dairy herds may partially overcome the antagonistic relationship between milk production and reproduction. Physiological mechanisms that lead to poorer reproduction in high producing cows are partially defined. Negative energy balance that occurs in high producing dairy cows can be associated with a delay in the initiation of ovarian cycles and the interval to first breeding. Many of the effects of negative energy balance on postpartum reproduction can be explained by decreased serum luteinizing hormone (LH) that is associated with negative energy balance. Serum LH increases as cows move toward positive energy balance and greater LH stimulates growth and ovulation of ovarian follicles. We have initiated studies to address physiological differences in high and low index dairy cows. The reproductive endocrinology of cows from a control line (5,900 kg milk/lactation) and a select line (10,900 kg milk/lactation) of dairy cows at the University of Minnesota was studied over a two-year period. Cows in Year 1 were similar for serum concentrations of LH, follicle stimulating hormone (FSH), and oestradiol (preovulatory period). In both years, serum concentrations of progesterone during luteal phases, however, were decreased in select cows. The Year 2 cows also had a delay in the return to oestrous cyclicity that was associated with reduced LH. The possibility that decreased progesterone causes infertility in dairy cows will require further study. Collectively, these data suggest that changes in blood progesterone concentrations may explain, partially, lower fertility in high index dairy cows.

Insulin-like growth factor binding proteins in the bovine anterior pituitary.

Insulin-like growth factor binding proteins (IGFBPs) were characterized in bovine anterior pituitary tissue, pituitary conditioned media, and serum collected during the preovulatory and early luteal phases of the estrous cycle. Effects of in vitro treatments of pituitaries with luteinizing hormone-releasing hormone (LHRH), estradiol, and progesterone on IGFBP secretion were also evaluated. Predominant IGFBPs detected in anterior pituitary tissue by immunoprecipitation, ligand blotting, and Northern blotting were IGFBP-5 (29 kDa), IGFBP-2 (32 kDa), and IGFBP-3 (36 and 39 kDa doublet). Conditioned culture media contained IGFBP-5, a slightly larger form of IGFBP-2 (33 kDa), the 36- and 39-kDa forms of IGFBP-3, and a more extensively glycosylated form of IGFBP-3 (44 kDa). In serum, IGFBP-5 was not readily detected, and IGFBP-3 (40- and 44-kDa doublet) and IGFBP-2 (34 kDa) were larger than in pituitary tissue. Levels of IGFBP-2, -3, and -5 in pituitary tissue decreased during the preovulatory period and were lowest in the early luteal phase. Treatment with LHRH increased IGFBP-2 levels in media twofold. Estradiol or progesterone did not alter IGFBP secretion in vitro. Predominant IGFBPs produced and released by anterior pituitary tissue were IGFBP-2, -3 and -5. The activity of IGFBPs fluctuates in the pituitary in association with changes in stage of estrous cycle, implicating IGFBPs as potential regulators of gonadotrope function.

Reproductive performance of common dentex, Dentex dentex, broodstock held under different photoperiod and constant temperature conditions

The effect of photoperiod and temperature on the timing and the quality of spawning, and on associated endocrine changes in circulating 17β-oestradiol, 11-ketotestoserone (11-KT) and vitellogenin (Vtg) were investigated in common dentex, Dentex dentex, undergoing their second reproductive cycle. The possibility was also explored of using the measurement of steroids in the culture water of broodstock tanks, rather than in individual blood plasma samples, as a potentially useful tool for assessing the physiological state of a fish without disturbing them. One group of fish was exposed to a simulated natural seasonal cycle and ambient temperature (CONTROL). The other two groups were exposed to simulated seasonal photoperiod cycles (12 month-long) but which were phase-shifted either three months before (ADVANCED) or after (DELAYED) the natural cycle. Temperature was kept at 19.4±0.9 °C all-year-round. In the CONTROL, spawning started in mid-April and lasted until mid-June, while in the ADVANCED group, spawning started 4 months earlier and in the DELAYED group 2 months later than the CONTROL. The total egg production, egg quality, hatching rate, relative fecundity, and spawning index of the experimental groups were similar to the controls. The differences in spawning time induced by photothermal manipulation were associated with a difference in the timing of peak concentrations of plasma E2, 11-KT and Vtg. In all three groups, the amounts of conjugated 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and free and sulfated 11-KT which could be extracted from the water during the spawning period were significantly higher than those found in the preovulatory period. However, the differences were mostly less than 2-fold suggesting that, at least under the conditions employed in this study, the method was of limited use for non-intrusive detection of gonadogenesis and spawning (as had been hoped). The observed differences in spawning time and in the seasonal changes of sex steroids and Vtg confirm and extend the findings on marine fish.

F29Vascular network in LUF syndrome

BackgroundThe principal aim was to reveal additional information and comprehension's on physiology of the normal and pathophsysiology of disturbed cycles, that may lead towards the new diagnostic approaches and treatments in infertile patients.MethodThe group of 22 patients included women with LUF syndrome. The emphasis was put on the intraovarian vascularity and especially on vascularity from inner vessels of ring of the follicle. Scans were performed on 5th, 10th, 12th, 21st and 24th day of menstrual cycle. A control group of 38 patients with regular menstrual cycles was selected on the basis of the normal gynecological, sonographical and hormonal findings. These patients had normal ovulatory cycles but were investigated for infertility due to the male factor.ResultsDoppler studies of the uterine artery and its evaluated branches have suggested significant decrease of the velocities at 12th and 15 h day of MC compared to control group. The RI of ovarian artery were around 0.84 ± 0.016 during the MC and excepted lowering of the RI has not occurred during the preovulatory period as in the control group. Velocity of ovarian artery of the dominant side showed significantly lower values, with the mean values of 34 cm/s ± 1.1 in the patients with LUF sy. compared to controls. The development of the RI of the intraovarian stromal vessels in LUF sy. were similar to that of anovulatory cycles, showing the loss in the cycling rhythm and post peak LH values similar to those of the follicular growth phase.ConclusionThe results of this study showed again that there is clear relationship between hormone mediators, primarily the steroids and velocimetric indices taken during the observed cycles. It also would seem that the hormones, with its mediators and hemodynamic factors play an important role through systemic and local action, in regulating uterine and ovarian blood flow.

Circadian rhythm changes in core temperature over the menstrual cycle: method for noninvasive monitoring.

The purpose of this study was to determine whether core temperature (T(c)) telemetry could be used in ambulatory women to track changes in the circadian T(c) rhythm during different phases of the menstrual cycle and, more specifically, to detect impending ovulation. T(c) was measured in four women who ingested a series of disposable temperature sensors. Data were collected each minute for 2-7 days and analyzed in 36-h segments by automated cosinor analysis to determine the mesor (mean temperature), amplitude, period, acrophase (time of peak temperature), and predicted circadian minimum core temperature (T(c-min)) for each cycle. The T(c) mesor was higher (P < or = 0.001) in the luteal (L) phase (37.39 +/-0.13 degrees C) and lower in the preovulatory (P) phase (36.91 +/-0.11 degrees C) compared with the follicular (F) phase (37.08 +/-0.13 degrees C). The predicted T(c-min) was also greater in L (37.06 +/- 0.14 degrees C) than in menses (M; 36.69 +/- 0.13 degrees C), F (36. 6 +/- 0.16 degrees C), and P (36.38 +/- 0.08 degrees C) (P < or = 0. 0001). During P, the predicted T(c-min) was significantly decreased compared with M and F (P < or = 0.0001). The amplitude of the T(c) rhythm was significantly reduced in L compared with all other phases (P < or = 0.005). Neither the period nor acrophase was affected by menstrual cycle phase in ambulatory subjects. The use of an ingestible temperature sensor in conjunction with fast and accurate cosinor analysis provides a noninvasive method to mark menstrual phases, including the critical preovulatory period.

Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system.

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.

Mechanism of delayed ovulation in a vespertilionid bat, scotophilus heathi: role of gonadotropin, insulin, and insulin-like growth factor-1.

The aim of this study was to evaluate the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor-1 (IGF-1) on ovarian androstenedione synthesis to understand the mechanism responsible for delayed ovulation in Scotophilus heathi. We found that LH stimulated a dose-dependent increase in androstenedione synthesis by the ovary in vitro. This study also showed a clear seasonal variation in the ability of the ovary to produce androstenedione in vitro in response to LH and FSH stimulation. In response to LH and FSH, maximum quantities of androstenedione were produced during recrudescence in November. The same doses of gonadotropins during the preovulatory period in February stimulated comparatively low androstenedione secretion by the ovary. On the basis of these data, we suggest that in S. heathi, ovarian responsiveness to LH and FSH peaks during recrudescence. This study also showed a seasonal variation in the effects of insulin and IGF-1 on ovarian androstenedione production in vitro. Peak ovarian responsiveness to insulin and IGF-1 was observed during quiescence in September. It is hypothesized that increased insulin/IGF-1 sensitivity during September may be responsible for increased responsiveness to LH. Increased LH release, if coincident with the period of enhanced ovarian responsiveness to LH, may result in the excessive androstenedione production responsible for delayed ovulation in S. heathi.

Follicle selection in cyclic guinea pigs with active immunization against inhibin α-subunit

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine α-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3±0.3 to 7.0±1.6 per each ovary), and preovulatory sized follicles (from 1.8±0.6 to 7.0±1.6 per each ovary), and follicles stained positively for inhibin α-subunit (from 2.3±0.5 to 6.3±1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin α-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.

Annual Cycles of Steroid Hormone Production, Gonad Development, and Reproductive Behavior in the Atlantic Stingray

The mating season of the Atlantic stingray (Dasyatis sabina), which begins in August and continues through April, is the longest documented for any elasmobranch fish. Despite this protracted mating period, female stingrays ovulate synchronously at the end of the mating season and there is no evidence for sperm storage by females. Thus, the proximate causal factors and ultimate function of this extended preovulatory mating are unknown. Annual cycles of the gonadal steroids testosterone (T), dihydrotestosterone (DHT), 17ß-estradiol (E2), and progesterone (P4) were measured for 26 months in a wild estuarine population of Atlantic stingrays to test for associations with their reproductive biology, gametogenesis, and sexual behavior. Serum androgen levels in males showed four phases within an annual cycle: (1) androgen suppression between reproductive seasons (April–July), (2) primary androgen increase during the onset of spermatocyte development (August–October), (3) androgen decrease following maximum testis growth and spermatocyte development (November–December), and (4) secondary androgen increase during the peak of sperm maturation (January–March). Increases in male E2 and P4 were correlated with spermatocyte/spermatocyst formation, maximum testis weight, and the primary (but not secondary) androgen surge. We propose that the production of male androgens across the full seven-month preovulatory mating period promotes their aggressive reproductive behavior and drives the protracted mating season of this species. In females, serum T and DHT showed relatively brief increases near ovulation, whereas E2 and P4 showed brief increases near both ovulation and parturition. The increase in female androgens near ovulation may increase female aggression when they are impregnable by courting males and enhance their choice of mates. This estuary sample population shows higher absolute steroid levels and distinct differences in temporal cycles compared to another Florida fresh water lake population, but the cause and significance of these differences are unknown. Experiments are needed to confirm that the aggressive and protracted mating behavior is the result of prolonged male androgen production and to determine whether the sustained preovulatory mating serves some function related to female reproduction.

Relationship between endometrial estrogen and progesterone receptors, and sonographic endometrial appearance in the preovulatory phase.

The purpose of the present study is to evaluate the relationship between endometrial concentrations of estrogen receptor (ER) and progesterone receptor (PR), and sonographic endometrial findings in the preovulatory phase of menstrual cycle. In 45 cycles of 45 infertile women with tubal factor only, transvaginal sonographic assessments and biopsy for immunohistochemical staining of the endometrium were made in the preovulatory phase of unstimulated, normal menstrual cycle. Immunohistochemical localization of ER and PR was scored according to intensity of staining and proportion of cells specifically stained in glandular epithelium and stroma, and the results were analysed according to the sonographic endometrial thickness (< 6 mm, 6-10 mm, or > 10 mm) and patterns. Endometrial patterns were classified as A, centrally hyperechogenic triple-line pattern or non-A, not triple-line. There were no significant differences in the endometrial thickness, serum estradiol level and serum progesterone level between A and non-A groups. The receptor scores of epithelial and stromal ER and epithelial PR were comparable in A and non-A groups. However, the receptor score of stromal PR was significantly higher in A group, with 4.8 +/- 1.4 compared with 2.7 +/- 1.7 in non-A group (p < 0.001). There were no differences in the receptor scores of epithelial ER, epithelial PR, stromal ER and stromal PR among the 3 groups according to the endometrial thickness. This study suggests that high PR expression in endometrial stroma could be related to the sonographic triple-line or multilayered pattern of endometrium in the preovulatory period.

Paracrine actions of oocytes in the mouse pre-ovulatory follicle.

In mammals, ovulation requires a tight control of extracellular matrix modifications, within both the follicle wall and the inner mass of granulosa cells surrounding the oocyte, namely the cumulus cells. During the pre-ovulatory period, mural granulosa cells promote selective degradation of perifollicular matrix, resulting in the formation of a follicle rupture site. Conversely, cumulus cells synthesize a large amount of a muco-elastic matrix that plays an essential role in the extrusion of the oocyte from the follicle and in the subsequent fertilization process. Formation of such matrix by cumulus cells in the pre-ovulatory follicle appears to be controlled by a paracrine influence by the oocyte. We have shown that mouse oocytes modulate the response of cumulus cells to an ovulatory gonadotropin stimulus by promoting the synthesis and preventing the degradation of cumulus matrix. Therefore, although gonadotropins are essential for triggering the complex events involved in ovulation, the oocyte appears to have an active role in this process. In the present review current data and hypotheses concerning molecular mechanisms involved in the organization and synthesis of cumulus matrix are discussed.

The physiological role of beta-endorphin in porcine ovarian follicles.

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.