Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.     

Cyclic AMP and steroidogenesis in superovulated rat ovaries

During the gonadotropin stimulated differentiation of ovarian follicles into corpora lutea, the concentrations of total cyclic AMP and protein bound cyclic AMP increased only marginally. In contrast, there was a 10 fold increase in progesterone production. After acute stimulation of luteinized ovaries with choriogonadotropin total cyclic AMP levels increase more than 6 times while bound cyclic AMP and plasma progesterone rose by 80 and 60% respectively. Our results question the role of cyclic AMP in the basal production of progesterone by the ovary, and suggest a relationship between bound cyclic AMP and progesterone synthesis only exists after acute gonadotropin stimulation.     

Secretion of pregnane compounds from follicular and luteal ovaries in rats

Differences during the follicular and luteal phases in the secretion of pregnane compounds by rat ovaries were studied. Daily s.c. injections of 2 mg of progesterone that began at early diestrus prolonged the diestrus stage for the duration of the treatment. In the follicular phase, normal proestrus and proestrus delayed by progesterone treatment were examined. In the luteal phase, day 6 of pseudopregnancy was examined. 20 alpha-hydroxy-4-pregnen-3-one accounted for most of total amount of pregnane compounds secreted from ovaries in the follicular phase, and progesterone and 20 alpha-hydroxy-4-pregnen-3-one for most at the luteal phase. The levels of 5 alpha-pregnane compounds were low in both phases. Injection of 2 micrograms of luteinizing hormone (LH) before sample collection increased the secretion of progesterone in the follicular phase, but not in the luteal phase. The secretion of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one was increased by the injection of LH in either phase, but the ability of the ovaries to produce these steroids was low, suggesting that there was low 5 alpha-reductase activity in the ovaries of rats with delayed proestrus and pseudopregnant rats.     

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.     

Response related enzymatic changes in ovaries of superovulated mice

Adult female mice were superovulated with PMSG followed by HCG and 140 blastocysts and 69 morulae were recovered from 24 mice. On the basis of the response, mice were divided into six groups; non responders, 1-5, 6-10, 11-20, 21-30 and >30 embryos. The ovaries of the animals were pooled group wise, homogenized in PBS (pH 7.4) and after centrifugation for 10-15 minutes, the supernatant was analyzed for the enzymes, guanine oxaloacetate transaminase (GOT), guanine pymvate transaminase (GPT), acid phosphatases (ACP) and alkaline phosphatases (AKP). Acid and alkaline phosphatase activities did not show any variation in relation to response to superovulation but GOT and GPT showed significantly increased activity in response to induction of superovulation. A statistically significant positive correlation was found between GOT and GPT activities and the superovulatory response in mice.     

Regulation of follicular development by diethylstilboestrol in ovaries of immature rats

Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.     

Long-term adverse effects of cyclophosphamide on follicular growth and angiogenesis in mouse ovaries

The adverse effects of the anti-cancer agent cyclophosphamide (CTX) on follicular growth and ovarian angiogenesis were investigated in mice. CTX treatment irreversibly induced a loss of follicles through apoptosis and decreased microvascularization of the corpora lutea and follicles in a dose-dependent manner. Our findings demonstrated that CTX adversely affected the ovaries indicating the need to support an awareness of fertility preservation before chemotherapy is initiated.     

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.     

Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish

Aiming to better understand folliculogenesis, this study evaluated cell death and proliferation of ovarian cells, besides cathepsin-D expression in Prochilodus argenteus captured in two sites of the São Francisco River downstream from the Três Marias Dam, Brazil. In the site immediately following the Dam (S1), low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower cells in proliferation and GSI, as well as a lesser quantity of cathepsin-D when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. Our results showed that apoptosis, cell proliferation and cathepsina-D evaluation can be used as biomarkers of environmental impact.     

Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers

A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)-stimulated (superovulated) and unstimulated heifers. Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol-17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy. The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid-mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH-stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley-Liss, Inc.     

Scaling of follicular sizes in mammalian ovaries

The scaling of ovarian follicle and oocyte sizes according to body weight ( M , ranging from 0005–500 kg) has been analysed using data obtained from 22 mammalian species in nine orders. The diameters of non-growing (primordial) follicles were correlated significantly with body weight, the relationship being described by the allometric formula y = 0028 M ^0.10. The mean size at which growing follicles began to accumulate extracellular fluid was approximately the same in all species, 0–3 mm diameter. Graafian follicle sizes varied allometrically with body weight as a result of differences in the volumes of follicular fluid rather than those of oocytes, which were relatively similar in eutherian mammals. The statistical significance of the correlation between Graafian and body sizes was increased when the dimensions for an ovulatory quota of follicles were combined because follicles in polyovulating species were disproportionately small. The total Graafian surface areas and volumes were then predicted from body weight by 58–4 M ^0.65 and 18–5 M ^1.06, respectively. Follicular dimensions in the three species of primates were significantly greater than predicted by the allometric relationship. The exponents of these relationships show that the total volume of a set of preovulatory follicles varies approximately isometrically with body weight and, therefore, with the presumptive hormone distribution volume ( M ^1.0). The hypoallometric relationship of follicular surface area demonstrates that, during the course of the evolution of body size, the surface area for secretion has not increased to match the dilution of hormones in the body pool.     

The effect of calcium on cholesterol side-chain-cleavage enzyme in superovulated rat ovaries

Cholesterol side-chain-cleavage enzyme activity in mitochondria isolated from heavily luteinized rat ovaries was determined using isocitrate as an electron donor and [4-14C]cholesterol to start the reaction. The activity of the enzyme was reduced when more than 10 microM calcium was added to the mitochondrial preparations. When 100 microM EGTA or 2 mM ATP was added to the reaction an increase in enzyme activity was observed and ATP was able to partially overcome the calcium-induced inhibition.     

Seasonal variation in follicular dynamics of superovulated Indian water buffalo

The ovaries of 5 buffalo were examined daily by ultrasound beginning at Day 3 of the estrous cycle, followed by superovulation between Days 11 and 13 of the cycle in both the wet cool and dry hot seasons. Daily ultrasonographic observations of the ovaries were recorded on a videotape and were used to assess the progression of both the large (dominant) and the next to the large (sub-dominant) follicles as well as the numbers of follicles in the small (4 to 6 mm), medium (7 to 10 mm) and large (> 10 mm) size categories in the 2 seasons before and during the superovulation treatment. Greater numbers of small (P < 0.05) and medium size follicles (P < 0.01) were available before the start of the superovulatory treatment in the buffalo during the dry hot season. The turnover of follicles from medium to large size classes also occurred sooner (P < 0.01) and was of higher magnitude (P < 0.05) during the treatment in the dry hot season. However, the number of corpora lutea at palpation per rectum (2.8 +/- 0.7 vs 2.2 +/- 0.6), the serum progesterone concentration (1.6 +/- 0.3 vs 1.4 +/- 0.1 ng/ml), and the yield of embryos on Day 6 (0.2 +/- 0.2 vs 0.6 +/- 0.2) did not differ significantly between the dry hot and the wet cool season. None of the embryos recovered during the dry hot season were transferable, which remains unexplained.     

Changes in follicular blood flow and nitric oxide levels in follicular fluid during follicular deviation in cows

Two experiments were conducted to test the hypothesis that there are dynamic changes in follicular blood flow during follicular deviation and that nitric oxide (NO) in follicular fluid (FF) plays a role in regulation of follicular blood flow. In Experiment I, follicular blood flow of the two largest follicles was monitored by using Power Doppler ultrasonography during follicular deviation in sixteen follicular waves during eight estrous cycles in eight cows. Blood flow did not differ (P>0.05) between the dominant follicle (DF) and the largest subordinate follicle (SF) until the beginning of the deviation of the follicular size, but was higher (P<0.05) in DF than in the largest SF one and two days after the beginning of diameter deviation in ovulatory (n=5) and atretic (n=11) waves; respectively. In Experiment II, FF was aspirated from DF and the largest SF on the day of diameter deviation (DF, n=6; SF, n=6) and two days later (DF, n=12; SF, n=9). Nitric oxide did not differ (P>0.05) between DF and the largest SF on the day of diameter deviation but, one or two days after observed diameter deviation NO concentrations were lower (P<0.01) in DF compared to the largest SF. On the day of diameter deviation and two days later E2 levels in FF were higher (P<0.01) in DF than in the largest SF. P4 concentrations in FF were higher (P<0.05) in DF than in the largest SF on the day of diameter deviation, but did not (P>0.05) differ two days later. E2/P4 ratio in FF was the same (P>0.05) in DF and the largest SF on the day of diameter deviation, but was higher (P<0.01) in DF than in the largest SF one or two days later. In conclusion, area of follicular blood flow of DF and the largest SF increased in parallel with follicular size during follicular deviation. Furthermore, there were relationships between changes in follicular blood flow, NO concentrations and E2/P4 ratio in FF following the beginning of diameter deviation in cattle.     

Cholesteryl ester depletion from the ovaries of superovulated female rats fed a normal or essential fatty acid deficient diet

The cholesteryl ester content of the ovaries was determined in rats diets containing corn oil or hydrogenated coconut oil (essential fatty acid (EFA) deficient) and subjected to superovulation by injection of luteinizing hormone and follicle-stimulating hormone. Superovulation increased ovarian weight; the effect was greater in animals fed corn oil. Superovulation significantly decreased total ovarian cholesteryl ester concentration in animals fed corn oil, with disproportionately large decreases occurring in the esters of 20:1, 20:2, 22:5w6, and 22:6w3. Significant decreases were observed in these esters when the data were expressed on a unit mass of tissue basis or in relation to total ovarian mass. In superovulated, EFA-deficient rats, esters of 18:1, 20:1, 22:5w6, and 22:6w3 were significantly lower per unit mass of tissue but this was due, in all cases except that of 22:6w3, to the increased mass of ovarian tissue; there was no decrease in total esters per ovary weight during superovulation of deficient rats. The pattern and degree of selective changes in ovarian cholesteryl esters during superovulation were different from those previously reported for adrenal esters of stressed rats.