Quantitative ultrastructural analysis of the follicular and luteal cells of the bovine ovary

Summary Ovaries were obtained from normal adult dairy cows at all days of the estrous cycle. The largest Graafian follicle and corpus luteum were excised, prepared for electron microscopic study, and their cell components quantitated using the linear scanning method and the counting of membrane crossings.The results indicated that in the theca interna cells during proestrus and estrus and in the large luteal cells during late metestrus and diestrus, enlarged mitochondria occupied an increased cytoplasmic percentage volume. During proestrus and estrus in the theca interna cells, the concentration of membranes of endoplasmic reticulum and Golgi apparatus also increased. The cytoplasmic percentage volumes of lipid bodies and of lysosomes increased in the small luteal cells; during luteal regression, they also increased in the large luteal cells. Similar rates of increase during follicular maturation, and decrease during luteal regression, occurred for measurements of succinic dehydrogenase and mitochondria.The quantitative observations were related to the production of steroid hormones by the ovary, and to the cyclic growth and regression of follicular and luteal cells. It was noted that an increased cytoplasmic percentage volume of mitochondria, an increased concentration of agranular cytoplasmic membranes, and low levels of lipid bodies and lysosomes, were generally present at times when ovarian steroid elaboration and cell growth were maximal.This investigation was supported by a General Research Support Grant to the College of Veterinary Medicine, University of Minnesota, and Research Grant No. GM-07009, of the United States Public Health Service. Approved for publication as Scientific Journal Series Paper No. 6343, Minnesota Agricultural Experiment Station. The work reported is taken from the senior author's Ph. D. thesis. Appreciation is expressed to Professor A.-M. CarPenter, Department of Anatomy, University of Minnesota, for her advice in matters concerning the quantitative techniques.     

The interaction of follicular cells and steroidogenic activity of the ovary

Investigations of ovarian cell types with the help of the tissue culture technique were presented. The importance of cell-to-cell communication and the interaction of the two cell types in producing steroid hormones by the ovary were discussed. Special attention was paid to the influence of prolactin on particular ovarian tissues. New concepts of the regulation of corpus luteum function were described.     

Role of gelatinase on follicular atresia in the bovine ovary

Follicular atresia, like follicular growth and ovulation, is characterized by excessive tissue remodeling. It is hypothesized that probably one of the tissue-remodeling enzymes, such as the gelatinases, could be playing an important role in this process. The present study was undertaken to determine the role of gelatinase on follicular atresia in the cow. Follicles of 2-6 mm in diameter were dissected from ovaries, and follicular fluid was categorized according to the morphological appearance of the cumulus-oocyte complexes. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography, and film in situ zymography was employed in order to localize gelatinase. TUNEL was performed on cryosectioned ovaries to understand follicular health. The concentrations of steroids in follicular fluid were also measured by solid phase fluoroimmunoassay. ProMMP-2 was detected in all normal and atretic categories of follicular fluid. The active form of MMP-2 and an additional band of proMMP-9 were detected only in atretic follicular fluid. Gelatinase activity was recorded in both granulosa cells (GCs) and theca cells (TCs) but were found in comparatively higher numbers in those follicles that exhibited a thinned and partially detached granulosa layer. TUNEL confirmed that apoptosis had commenced in the GCs of follicles of the latter category. The estradiol-17beta (E(2)):progesterone (P(4)) ratio was found to be significantly lower in atretic follicles than in normal follicles. These results suggest a plausible role for gelatinase in follicular health, especially the active form of MMP-2 and proMMP-9, and that bovine follicular fluid may be a key indicator of atresia.     

The succinic dehydrogenase of seedlings

Crystalline lysozyme has been interacted with an anionic, a cationic, and two nonionic surface-active agents (SAA). Quantitative precipitation of lysozyme by the ionic SAA used was obtained at ratios of the reactants consonant with the formation of stoichiometric complexes dependent upon salt linkages between the SAA and the oppositely charged groups in the enzyme. Neither of the nonionic SAA tested caused precipitation of the enzyme.The inactivation of lysozyme is shown to be constant over a 50-fold range of enzyme concentration when calculated on the basis of the ratio of SAA to enzyme. Inhibition of lysozyme activity as a result of interaction with ionic SAA was obtained only when the ionic SAA were present in substantial excess of the amount required for formation of stoichiometric complexes with oppositely charged groups in the enzyme. Neither of the two nonionic SAA studied altered the enzymatic activity of lysozyme.     

Comparison of the steroidogenic capacity of bovine follicular and luteal cells, and corpora lutea originating from dominant follicles of the first or second follicular wave

This study, compared the endocrine function of dominant follicles of the first and second follicular waves (DF1 and DF2, respectively) and the corpora lutea that were subsequently formed. In the experiments conducted in vitro, ovaries were collected from dairy cows on day 6.1 +/- 0.2 or day 14.8 +/- 0.2 of the oestrous cycle to obtain steroidogenically active DF1 (n = 8) and DF2 (n = 7). Granulosa and thecal cells were isolated, dispersed and incubated for 16 h with testosterone (granulosa cells) or forskolin or bLH (thecal cells). Both types of cell were subsequently cultured for 9 days with forskolin and insulin. The viability of the granulosa cells was similar in DF1 and DF2, but the concentration of oestradiol in the follicular fluid was higher in DF1 than in DF2. Production of oestradiol and progesterone by granulosa cells was similar in DF1 and DF2, but androstenedione and progesterone production by thecal cells were 3.5-6.5-fold higher in DF1 than in DF2. During the 9 days of luteinization, progesterone production was similar in DF1- and DF2-derived granulosa cells, but was two- to three-fold higher in DF1- than in DF2-derived thecal cells. Experiments were also conducted in vivo. In Expt 1 in vivo, lactating cows that were assigned to ovulate DF1 or DF2 (n = 9 and 13 in replicate 1 and 2, respectively) were injected with PGF2 alpha on days 6 and 7 or on days 14 and 15 of the oestrous cycle, respectively. A wave by replicate interaction was detected for plasma progesterone concentration in the subsequent cycle: in the first replicate, progesterone production was approximately 40% higher in cows that ovulated DF1; in the second replicate, progesterone production was similar in cows that ovulated DF1 or DF2. In Expt 2, pooled plasma progesterone in the mid-luteal phase (days 12-15) after insemination of pregnant and non-pregnant cows was approximately 30% higher in cows that had ovulated DF1 (n = 32) than in cows that had ovulated DF2 (n = 22). This study showed DF1 had a higher steroidogenic capacity compared with DF2, which may be related to the hormonal environment in which the follicles developed.     

Inhibition of progesterone synthesis and cAMP accumulation in small bovine luteal cells by a low molecular weight component of bovine follicular fluid

Follicular fluid from large follicles of cows was extracted with charcoal and filtered through an Amicon XM-50 membrane. The XM-50 filtrate was further fractionated on a column of Fractogel TSK HW-40 (s) using Krebs-Ringer-phosphate buffer (1/100th dilution), pH 7.2, as an eluant. Two fractions (1 and 2) were obtained. Inhibition of progesterone secretion by small luteal cells was associated with the XM-50 filtrate and Fraction 2. Whole follicular fluid, the XM-50 retentate and Fraction 1 had no significant inhibitory activity. Fraction 2, which contained about 1/100,000th of the original follicular fluid proteins, inhibited the LH- or (Bu)2cAMP-induced progesterone production during a 2-h incubation. This inhibition was dose-dependent. Fraction 2 also inhibited LH-induced cAMP accumulation, but did not affect the conversion of pregnenolone to progesterone or the basal progesterone production. The molecular weight of the inhibitory factor was estimated to be less than 10,000 and its ability to inhibit steroidogenesis was lost after digestion with protease but retained after heating for 60 min at 75 degrees C. These results demonstrate that bovine follicular fluid contains a heat-stable factor likely to be a polypeptide and which suppresses the steroidogenic response of small luteal cells to LH. The action of this inhibitory factor could involve both an inhibition of the LH-induced synthesis of cAMP and an inhibition of the action of cAMP.     

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 microM). Acrolein potently inhibited SSADH activity (IC(50)=15 microM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC(50) of 110 microM. Trans-2-hexenal (HEX) and crotonaldehyde (100 microM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.     

GABA影响大鼠卵巢黄体细胞孕酮的生成

实验用离体培养方法观察ＧＡＢＡ对大鼠黄体细胞孕酮及羟自由基（．ＯＨ）生成的影响。结果表明：ＧＡＢＡ抑制黄体细胞孕酮的生成,同时也促进黄体细胞．ＯＨ的生成。ＧＡＢＡ对孕酮的抑制作用可能与腺苷酸环化酶系统及ＧＡＢＡＡ型受体有关,而与蛋白质合成无关。