Expression of proopiomelanocortin-related peptides in human follicular fluid

In order to evaluate the expression of the opioid precursor proopiomelanocortin (POMC) in the ovarian follicle, we measured 6 of its main end-products in 23 follicular fluids. We coupled high performance liquid chromatography (HPLC) to specific radioimmunoassays. Seven follicles were immature (diameter less than 9 mm), 10 were obtained from superovulated patients during an in vitro fertilization-embryo transfer program (greater than 22 mm) and six were persistent follicles, collected during the luteal phase [15-31 mm, luteinized unruptured follicles (LUF)]. Follicular fluids were extracted by mean of Sep-pak cartridges and then purified by HPLC with a reverse-phase C-18 column eluted in a linear gradient with acetonitrile/0.01 M hydrochloric acid (from 18:82 to 40:60). Fractions were tested with specific antisera for ACTH (1-39), alpha-MSH, beta-lipotropin (beta-LPH), beta-endorphin (beta-EP) and gamma-endorphin (gamma-EP) immunoreactivities. No presence of beta-LPH, beta-EP and ACTH was confirmed, while gamma-EP, alpha-MSH and des-alpha-MSH were detected for the first time in follicular fluid. In every class of follicles shorter chain peptides predominate over their longer chain precursor. Immature follicles are characterized by the highest amounts of gamma-EP, ACTH, alpha-MSH and des-alpha-MSH if compared to superovulated and LUF. On the contrary, beta-EP amount was highest after superovulation. Apart from this finding, peptide levels in superovulated patients and LUF are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis in granulose cells induced by intrafollicular peptide

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries has been earlier shown to induce degeneration of ovarian follicles in mice. In the present study, whether the effect of OFFP on granulosa cells was similar to apoptosis was studied using three parameters. Immature mice injected with pregnant mare serum gonadotropin on day 0 were administered with 10 or 20 μg of OFFP on day 1 and autopsied on day 2. The granulosa cells were collected from the ovarian follicles. The presence of apoptotic bodies were observed by staining the cells with acridine orange. DNA profiles of DAPI-stained cells analysed by flow cytometry also revealed apoptotic response to OFFP. Furthermore, agarose gel electrophoresis of low molecular weight DNA fraction extracted from the cells of OFFP-treated animals confirmed ladder formation and induction of apoptosis and not necrosis in granulosa cells. In conclusion, all the three parameters indicated apoptotic changes in granulosa cells of ovarian follicles in .mice treated with OFFP. The effect of OFFP seems to be exerted directly on the granulosa cells showing its autocrine role in the process of follicular atresia. This is discussed in the light of other intra/extra ovarian factors.     

Stimulatory and Inhibitory Effects of Human Follicular Fluid on Amphibian Oocyte Maturation and Ovulation hi vitro

Follicular fluid was collected from individual human ovarian follicles and its effects, alone or in combination with frog pituitary homogenate (FPH), on oocyte maturation and ovulation were assessed following incubation with amphibian ovarian follicles in vitro. Oocyte maturation, with little or no concomitant ovulation, was induced by variable amounts of follicular fluid. Some of the individual follicular fluid samples were very active in inducing oocyte maturation, whereas others were inactive. Frog pituitary homogenates exhibited biologic activity (induced oocyte maturation and ovulation) when incubated in the presence of most follicular fluid samples. However, follicular fluid samples from two individuals inhibited ovulation but not maturation in FPH-treated follicles. These results demonstrate that amphibian follicles remain viable and undergo a number of physiologic changes in the presence of unfractionated human follicular fluid. Under appropriate conditions both stimulatory and inhibitory effects of follicular fluid were observed. These data suggest that amphibian ovarian follicles may provide a simple and independent means for detecting and assaying a number of biologic activities present in follicular fluid obtained from single human and other mammalian ovarian follicles. Such results may provide the basis for dissociating endocrine and cellular interactions which occur during normal and abnormal follicular differentiation.     

Hamster oocyte maturation in vitro: Inhibition by follicular components

When prevulatory hamster follicles were cultured $\text{in vitro}$, the oocytes within them remained at the germinal vesicle stage. This maturation arrest was partly overcome by washing the follicles before cultivation or by the addition of LH to the medium. LH-reversible oocyte arrest was also induced in isolated oocytes by culturing them either with the cumulus oophorus or with hamster follicular fluid was not species-specific, inhibitory effect of follicular fluid was not species-specific, since an LH-reversible inhibition was also produced by follicular fluid of bovine origin. Evidence is presented indicating that the inhibition is due to a heat labile peptide with a molecular weight between 10 0 and 10,000. LH may induce oocyte maturation by acting on the oocyte so that it no longer responds to the inhibitor.     

Purification, measurement, and tissue distribution of a dansyl-derivatized glycopeptide from low-molecular weight follicle-stimulating hormone-inhibitor-containing fractions of porcine follicular fluid

We have used dansyl chloride (5-dimethylamino-1-naphthalenesulfonyl choloride) to form dansyl derivatives of amine-containing compounds in follicular fluid or highly purified fractions containing a low molecular weight (MW) inhibitor of follicle-stimulating hormone (FSH) binding to receptor (FSH-BI). This approach allowed sensitive detection of the derivatives based on their fluorescent properties. By taking advantage of the hydrophobic nature of the dansyl group, a dansyl derivative (RF = 0.15) identified in low MW FSH-BI preparations was purified from porcine follicular fluid. Based on chromatographic criteria using four different systems (thin-layer chromatography [TLC] and high performance liquid chromatography), the derivatized factor (D15) that was purified appeared to be homogeneous. A direct, chemical assay was developed for quantification of D15 from follicular fluid or tissue extracts. The highest concentration (153 ng/mg) of D15 was found in ovarian tissue of adult rats, lesser amounts were observed in kidney and liver tissues (93 and 62 ng/mg, respectively) and even less in diaphram and heart tissues (5 and 0.5 ng/mg, respectively). High concentrations of D15 were observed in derivatized extracts of tests from immature rats in which approximately twice as much D15 was found in Leydig cells (241 ng/mg) as in seminiferous tubules (136 ng/mg). In porcine ovarian tissue, granulosa cells from large follicles and corpora lutea (69 and 91 ng/mg, respectively) contained at least 4-fold higher concentrations than follicle wall tissue (14 ng/ml). Relative concentrations of D15 material were also determined in pools of bovine follicular fluid previously shown to contain low MW FSH-BI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.     

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.     

Sex steroid changes in porcine cystic ovarian disease

Ten sex steroids were measured in the peripheral serum and ovarian follicular fluid of female pigs with or without cystic ovarian disease. In general, progestin, especially progesterone, accumulated excessively in the fluid contained in cystic compared with normal follicles. Nonluteinized cystic follicles contained up to four times the progesterone concentration found in large normal preovulatory follicles. Levels of this steroid increased with luteinization of cystic follicles to as much as 10 times those found in large preovulatory follicles. In contrast, the concentration of follicular fluid androgens and estrogens in cystic follicles were, at best, barely detectable (5 to 10 pg/ml). These results are indicative of a steroidogenic blockade in the conversion of C21 progestin to C19 androgens and C18 estrogens in the cystic follicles. In spite of an enormous accumulation of follicular progestin and subnormal concentration of androgens and estrogens, circulating levels of these hormones in pigs bearing cystic ovaries were in the normal range for cycling sows. Clearly, the hormonal abnormalities in the cystic follicles are not reflected in the serum profiles of these steroids.     

Relationship between intrafollicular concentrations of parathyroid hormone-related peptide (PTHrP) and steroid hormones in oestrogenic and non-oestrogenic ovarian follicles in the mare

The objective of the present study was to determine whether parathyroid hormone-related peptide (PTHrP) is present in the equine follicular fluid and if so, how it is related to the follicular development in the horse. For this purpose, ovaries were collected from 40 Thoroughbred and Thoroughbred Cross mares at slaughter during the period from February to May. Normal growing follicles were dissected from the ovaries of each mare and their diameters measured. A total of 174 follicles was used in this study. The follicular fluid was aspirated from each follicle and assayed for PTHrP, oestradiol (E), testosterone (T) and progesterone (P). The follicles were classified as either oestrogenic or non-oestrogenic if the follicular fluid content of oestradiol was >40 or <40 ng/ml, respectively. PTHrP concentrations were significantly (P<0.05) higher in oestrogenic follicles, but T and P concentrations did not differ. Furthermore, E:T ratio was significantly (P<0.05) greater in oestrogenic follicles compared to the non-oestrogenic ones. The mean diameter of oestrogenic follicles was significantly (P<0.05) greater than that of non-oestrogenic ones. The higher concentrations of PTHrP observed in the follicular fluid of healthy oestrogenic follicles suggest that it may have a role in the control of ovarian function.     

Sequence of apoptosis and inflammatory necrosis within the formative ovulatory site of sheep follicles

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.     

Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes

The objective was to determine the effect of dietary intake on follicle and oocyte morphology in unstimulated and superovulated ewes. Fifty-four ewes were fed grass meal at 0.5, 1.0 or 2.0 times maintenance energy requirements (M) for 32 days. Oestrous cycles were synchronized using progestagen pessaries and either unstimulated or superovulated with 200 mg pig FSH. The ewes were killed and ovaries were collected either 36 or 12 h before the anticipated LH surge. Serum progesterone concentrations in ewes on day 10 after withdrawal of the pessary were lower in ewes fed 2.0M than in ewes fed 0.5M or 1.0M (P < 0.05). LH pulse frequency tended to be higher in ewes fed 2M than 1M (1.0 +/- 0.3 versus 0.3 +/- 0.2 pulses per 8 h) on day 6 after removal of the pessary but the effect was not significant. In unstimulated ewes, more follicles (>/= 3 mm) were observed when the animals were killed in ewes fed 2.0M (3.5 +/- 0.3) than in ewes fed 0.5M (2.4 +/- 0.3) or 1.0M (2.4 +/- 0.5; P < 0. 05). Fewer follicles were observed in superovulated ewes on 0.5M (7. 5 +/- 1.2) than in ewes on 1.0M (12.0 +/- 0.5) or 2.0M (12.3 +/- 1. 4; P < 0.05). Follicular fluid progesterone concentrations were higher in ewes fed 0.5M compared with those fed 1M or 2M (P < 0.05). Insulin-like growth factor (IGF)-I concentrations were higher in follicular fluid from ewes on 1M compared with either those on 0.5M or 2M (P < 0.05), whereas IGF-II concentrations were lower in follicular fluid from ewes on 2M compared with those on 1M or 0.5M (P < 0.05). Superovulation increased follicular fluid progesterone, oestradiol, IGF-I and IGF-II concentrations (P < 0.01). Concentrations of the 34, 22 and 20 kDa IGF binding proteins were lower in follicles from superovulated ewes compared with unstimulated ewes (P < 0.05). Oocytes from superovulated ewes showed abnormalities such as premature activation of cumulus expansion and vacuolation of the nucleolus and increased frequency of detachment of interchromatin-like granules from the nucleolar remnant. Collectively, these results indicate that both high and low dietary intakes can alter systemic and follicular fluid hormone concentrations. Relative to dietary effects, the effects of superovulation were greater and involved substantial increases in follicular fluid hormone concentrations and abnormal oocyte morphology.     

Ovarian activity in synchronized mares following administration of follicle stimulating hormone

Twelve non-pregnant, non-lactating, light horse type mares of unknown breeding were randomly assigned to two treatment groups with six replicates per group. Mares were administered PGF_2α (10 mg, IM) on days 0 and 14, and HCG (3000 IU, IM) on days 6 and 20. Group A received FSH (5 units, IM) twice daily (06.00 and 18.00 h) on days 15 through 10. Group B received saline twice daily (06.00 and 18.00 h) on days 15 through 19. Ovaries were recovered at necropsy on day 30 (10 days post-ovulation). Ovaries were weighed, CL number and weight determined, follicles counted and measured, and volume of follicular fluid quantified.Mean ovarian weight (g) and number of CL per mare, respectively, were: Group A, 148.1 ± 62.3, 0.83 ± 0.31; Group B, 91.3 ± 16.8, 0.83 ± 0.81. Mean number of follicles > 10 mm and total volume (ml) of follicular fluid per mare, respectively, were: Group A, 2.25 ± 0.71, 38.7 ± 26.3; Group B, 2.25 ± 0.73, 14.7 ± 4.9. There was no difference (P > 0.05) in mean ovarian weight, CL number, CL weight, follicular fluid volume, number of follicles, or size of follicles between treatment groups. These results show no significant effect on ovarian activity in mares following administration of exogenous FSH.     

Biochemical composition of ovine follicular fluid in relation to follicle size

The aim of this study was to biochemically characterize ovine follicular fluid and to relate possible changes in composition to follicular size. Ovaries were collected from adult and cycling non-pregnant slaughtered sheep (Ovis aries) during breeding season. A total of 104 pairs of ovaries were investigated and these data were then compared. Follicular fluid was aspirated from small (< 2 mm), medium (2-4 mm) and large (> 4 mm) nonatretic ovarian follicles. The follicular fluid was centrifuged at 4 degrees C and 5000 g for 30 min to remove any cells and stored at -80 degrees C prior to assay. Follicular fluid samples were analyzed for glucose, total protein, cholesterol, triglycerides, lactate, urea, creatinine, sodium, potassium, chloride, calcium, phosphorus, magnesium, acid phosphatase, alkaline phosphatase, and lactate dehydrogenase. Data were analyzed by the linear regression model. As follicles became larger, the concentrations of glucose and cholesterol significantly (P < 0.05) increased while those of triglycerides, lactate, alkaline phosphatase and lactate dehydrogenase significantly (P < 0.05) decreased.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: a mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles in vitro and found that hFF1 contains a factor of high molecular weight (Mr greater than 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr less than 1000) which stimulated PG synthase activity. If this is true of ovarian follicles in vivo, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: A mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles and found that hFF contains a factor of high molecular weight (Mr > 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr < 1000) which stimulated PG synthase activity. If this is true of ovarian follicles , it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Preovulatory endocrinology and oocyte maturation in superovulated cattle

Preovulatory follicular and oocyte nuclear maturation was studied in donor cattle induced to superovulate with a PMSG- or FSH-prostaglandin regimen. Plasma concentrations of progesterone (P4) and luteinizing hormone (LH) and follicular fluid levels of progesterone and estradiol-17β were measured and related to the oocyte nuclear maturation stages. The oocyte donors could be divided into two distinct groups. Group I had entirely normal periovulatory P4 and LH concentrations, and the majority of follicles and oocytes followed a characteristic pattern, clearly time-related to the LH peak. Group II had deviating levels of P4 and/or LH in the pre- and periovulatory period, and a majority of their follicles and oocytes had disturbed maturation, such as abnormal P4:E2 ratio in follicles and prematurely activated or meiotically arrested oocytes. It is concluded that a certain proportion of superovulated cows and heifers develop abnormal follicular/oocyte maturation and constitute poor oocyte, and probably also embryo, donors.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats

Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day--a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days--a time when the earliest signs of impending follicular destruction is seen, 15 days--a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 +/- 0.14 for 10 days, 1.48 +/- 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 +/- 0.11 for 10 days, 1.37 +/- 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0.29 +/- 0.04; 15 days, 0.51 +/- 0.04, VCD/control, P < 0.01). VCD did not affect any of these measurements in large preantral follicles or liver. Phosphorylation status of c-Jun protein as measured by Western blotting was increased (1.22 +/- 0.1, VCD/control, P < 0.05) after the 15-day daily dosing with VCD, but total c-Jun protein levels were unaffected. Using antibodies against c-Jun or phospho-c-Jun for supershift DNA binding assay, c-Jun and phospho-c-Jun were identified in the AP-1-DNA binding complex, and the binding activity was reduced in tissues with increased phospho-c-Jun protein levels. Taken together, these data provide evidence that accelerated atretic signals induced by VCD is associated with MAPK/AP-1 signaling pathways and phosphorylation of c-Jun plays a significant role in transmitting the apoptotic signals.