The presence of luteinized unruptured follicle syndrome and altered folliculogenesis in rats with surgically induced endometriosis

OBJECTIVE: Our purpose was to determine in the rat model whether endometriosis could influence ovarian function by altering oocyte release or folliculogenesis. STUDY DESIGN: We histologically examined the ovaries of reproductively cycling rats with (n = 16) and without (n = 10) surgically induced endometriosis. The rats in these two groups were further subdivided into unilaterally ovariectomized or ovarian-intact groups. Serial sections of ovaries were examined, and follicular development and frequency of luteinized unruptured follicles were determined. RESULTS: A significant tenfold increase in the number of luteinized unruptured follicles was observed in the ovaries from rats with endometriosis (2.7 per rat) compared with unoperated and sham-operated control groups (overall mean 0.26 per rat, p < 0.05). Additionally, ovaries from unilateral ovariectomized animals with endometriosis contained four times as many luteinized unruptured follicles (four per rat) as did the ovaries from bilaterally ovarian-intact rats with endometriosis (1.40 per rat, p < 0.01). Fewer follicles were present in rats with endometriosis (180 follicles per ovary) than in control rats (231 follicles per ovary, p < 0.05). CONCLUSION: In the rat model the presence of ectopic endometrium is associated with an increased frequency of luteinized unruptured follicles and altered follicular development.     

Calcium concretions in the pineal gland of aged rats: an ultrastructural and microanalytical study of their biogenesis

OBJECTIVE: We summarized general principles related to apoptosis and reviewed the current evidence regarding apoptosis in the normal ovary during follicular atresia, ovulation, and corpus luteum formation and regression, and in the polycystic ovary. DATA SOURCES: Published articles and abstracts from national scientific meetings were reviewed. DATA SYNTHESIS: Apoptosis is a physiologic type of cell death that may regulate tissue remodeling. In the ovary, apoptosis occurs in atretic follicles, in the ovarian epithelium at the time of ovulation, and in the corpus luteum. Certain apoptosis-related genes are expressed during follicular atresia, which suggests a role for genes in the regulation of apoptosis in the ovary. In a rat model of polycystic ovaries, apoptosis occurs in both atretric follicles and cysts. CONCLUSIONS: Apoptosis is an important physiologic process in the ovary, and abnormalities in this process may lead to pathologic conditions. The study of ovarian apoptosis is still at an early stage, and further research will uncover the steps in this process.     

Differential expression of estrogen receptor-beta and estrogen receptor-alpha in the rat ovary

Immunohistochemical localization of two estrogen receptor (ER) subtypes, ER beta and ER alpha, was performed in neonatal, early postnatal, immature, and adult rats to determine whether ER alpha and ER beta are differentially expressed in the ovary. ER beta and ER alpha were visualized using a polyclonal anti-ER beta antibody and a monoclonal ER alpha (ID5) antibody, respectively. Postfixed frozen sections and antigen-retrieved paraffin sections of the ovary revealed nuclear ER beta immunoreactivity (IR) in granulosa cells, which was prevented when peptide-adsorbed antibody was used instead. In immature and adult rat ovaries, ER beta was expressed exclusively in nuclei of granulosa cells of primary, secondary, and mature follicles. Atretic follicle granulosa cells showed only weak or no staining. No specific nuclear ER beta IR was detected in thecal cells, luteal cells, interstitial cells, germinal epithelium, or oocytes. In neonatal rat ovary, no ER beta expression was found. In ovaries of 5- and 10-day-old rats, weak ER beta IR was observed in granulosa cells of primary and secondary follicles, but no staining was detected in the primordial follicles. ER alpha protein exhibited a differential distribution in the ovary with no detectable expression in the granulosa cells but evidence of ER alpha IR in germinal epithelium, interstitial cells, and thecal cells. In the oviduct and uterus, IR for ER alpha, but not ER beta, was found in luminal epithelium, stromal cells, muscle cells, and gland cells. Our present study demonstrates that ER beta and ER alpha proteins are expressed in distinctly different cell types in the ovary. The exclusive presence of ER beta in granulosa cells implies that this specific new subtype of ER beta mediates some effects of estrogen action in the regulation of growth and maturation of ovarian follicles.     

Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.     

The normal development of the ovary in childhood

The development of the ovary during childhood has been evaluated in 52 organs obtained from autopsy of children who died in accidents or after a brief acute disease. The ovaries at all ages showed follicle growth. 93% of the organs were actively growing ovaries containing healthy and degenerating follicles in progressive stages of development with and without follicle fluid. Three ovaries showed follicle growth up to pre-antral stages. None of the 52 ovaries were quiescent in which follicle development was not in progress. It is therefore argued that the normal ovary in childhood shows follicle growth at all ages. The number and size of antral follicles increases after the age of 6 years coinciding with the progressive increase in FSH and oestrogen output in childhood.     

The ovary. Computed tomography and magnetic resonance imaging

CT and MR imaging can both contribute valuable clinical information in women with benign and malignant ovarian masses; the superior soft tissue contrast and multiplanar capabilities provided by MR imaging make it a valuable tool to evaluate the normal ovary, polycystic ovaries, endometriosis, and disorders of sexual differentiation. CT is currently the recommended modality to stage ovarian carcinoma, and peritoneal implants as small as 5 mm can be visualized. With the addition of contrast-enhanced images to conventional MR sequences, however, improved visualization of intratumoral architecture has also allowed for accurate MR characterization of benign versus malignant ovarian masses. Fat saturation techniques used with conventional MR sequences can also be used for definitive characterization of benign fat-containing teratomas and differentiate these tumors from hemorrhagic ovarian cysts or endometriomas. Finally, the detailed visualization of the normal-sized ovary on MR images provided by both the body coil and the new phased-array coil allows detection of the normal ovaries, ovarian follicles, and ovarian cysts throughout the premenopausal and postmenopausal years. Findings of polycystic ovarian syndrome, which cannot be evaluated by CT, are characteristic on MR images. MR is unparalleled in the evaluation of disorders of sexual differentiation because MR images can depict ectopic and normally positioned gonads and provide multiplanar depiction of the other pelvic organs and perineum as well. CT and MR imaging can provide valuable and specific clinical information about the ovary and benign and malignant ovarian disease.     

Cell adhesion molecules and pharmacologic applications. Round Table No 3 at Giens XIII]

The mature ovary contains a large number of macrophages. In the present study, the distribution of macrophages in murine ovaries at various developmental stages was immunohistochemically studied using a monoclonal antibody against F4/80, a highly specific antigen of murine macrophages. The results showed that definite F4/80-positive stains were hardly detectable in ovaries on day 0 after birth. On day 7, a few F4/ 80-positive cells could be identified between the developing follicles. The positive stains were irregular in shape and showed little physical contact with the primordial or primary follicles. By days 14 and 21, when the theca cell layers of growing follicles were developing, the positive cells had extended or elongated to surround the cell layer. On day 28, besides the presence of elongating positive cells surrounding the growing follicles, irregularly shaped F4/80-positive cells became apparent in the interstitium between the growing follicles and also in the capsular tissues. Thereafter, positive cells with stellate appearance were detected in the corpora lutea, which first developed around 6 weeks of age. Although the positive cells were homogenously distributed in the corpora lutea in virgin adults, only a few sporadic positive cells were found there in pregnant mice. However, the positive cells infiltrated into the corpora lutea again in the postpartum period. These results show that ovarian macrophages exhibit dramatical changes in their distribution from neonatal to postpartum periods.     

Patterns of ovarian cell proliferation in rats during the embryonic period and the first three weeks postpartum

The purpose of this study was to characterize the patterns of ovarian cell proliferation during the earliest stages of folliculogenesis, which occur in the embryonic period and the first weeks postpartum in rats. Rats were given continuous infusions of [3H]thymidine (3H-TdR) or bromodeoxyuridine (BrdU), and cells that were synthesizing DNA were visualized by autoradiography or immunohistochemistry. There were dramatic changes in the patterns of cell proliferation during the period studied. Mesenchymal cells proliferated extensively in the embryonic and neonatal ovary, but their growth fraction declined precipitously as follicles formed. Epithelial cells in the medulla of the ovary left the cell cycle at about embryonic Day 12, then resumed proliferation as soon as they were incorporated into follicles just after birth. Epithelial cells towards the cortex of the organ continued to proliferate until late in the embryonic period; they apparently became quiescent around the time of birth, and incorporation into follicles did not release them from their quiescent state. After the follicles had formed, patterns of cell proliferation continued to change. At 5 days postpartum, approximately 36% of the smallest follicles (1-8 granulosa cells in cross section) had at least 1 granulosa cell that was labeled following a 24-h infusion of 3H-TdR; by Day 20 only 14% of these follicles were labeled, and by Day 30 only 4.4% were labeled.     

Immunocytochemical localization of vasoactive intestinal peptide and neuropeptide Y in the bovine ovary

The distribution of the neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) was studied immunocytochemically in bovine ovaries from 3 mo of gestation up to and including puberty, and from adult cows at three stages of the estrous cycle. The appearance of VIP and NPY immunoreactivity of 4.5-6 mo of gestation coincided with the onset of follicular development. In contrast to NPY, VIP was first found in the cortex. Both VIP and NPY immunoreactivity increased with age. From 9 mo of gestation onwards, VIP and NPY were found around blood vessels and non-vascular smooth muscle cells, in the stroma near preantral follicles, and in the theca externa of antral follicles. In addition, VIP-positive cells were observed exclusively in the granulosa layer of the preovulatory follicle at the time of the LH surge. The distribution of VIP- and NPY-immunoreactive fibers in the ovary may point to an effect of these neuropeptides on various physiological processes, including follicle development and ovarian blood flow. In addition, the presence of VIP-positive cells in the granulosa layer of the preovulatory follicle is indicative of a role for VIP in ovulation.     

Characterization of fetal ovine renal dysplasia after mid-gestation ureteral obstruction

OBJECTIVE: The etiology and pathogenesis of renal dysplasia are poorly understood. To characterize the histologic changes in fetal renal dysplasia, we studied a fetal ovine model of urinary obstruction. DESIGN: Animal study. ANIMALS: Seven fetal lambs, and other lambs of the same gestational age as controls. INTERVENTIONS: Unilateral ureteral ligation on fetal lambs at approximately 70 days' gestation (term for sheep is 145 days), during nephrogenesis. Kidneys were subsequently collected, examined histologically and characterized by immunohistochemical tests involving cytokeratin antiserum and a monoclonal antibody to alpha-actin. OUTCOME MEASURES: Histologic changes in ligated fetal lamb kidneys, based on comparison with normal fetal lamb kidneys. RESULTS: At near term (140 days' gestation), the ligated kidney showed distorted and less abundant renal parenchyma than a normal control kidney. Upon microscopic examination, the ligated kidney displayed marked architectural distortion of the outer cortex, with abundant interstitial fibrosis, primitive ductules and glomeruli, and cysts of varying sizes lined by squamous and cuboidal epithelia and surrounded by a loose mesenchyme. The renal medulla contained differentiated collecting ducts, which were structurally distorted and less abundant than in normal control kidneys. The proximal and distal tubule elements were primitive and markedly underdeveloped. Cytokeratin immunoreactivity was present in the collecting duct epithelium and in the cuboidal epithelium lining many of the cortical cysts. Smooth muscle alpha-actin immunoreactivity was localized in the cortical region of the kidney, which highlighted the abundance and disorganization of the undifferentiated mesenchyme and identified the fibromuscular collars of the primitive ductules of the cortex and the distorted collecting ducts of the medulla. CONCLUSIONS: These results highlight the histologic changes resulting from unilateral ureteral ligation in fetal lambs. This model is useful in the study of the pathogenesis of fetal obstructive renal dysplasia.     

Cardiological effects of catecholamine-secreting tumours

Ovaries (N = 250) from slaughtered buffaloes were collected to study follicular population and compare methods of oocyte retrieval. The number and size of surface follicles were recorded and grouped into different categories. Different sized follicles in relation to oocyte diameter were studied histologically. Yield of oocytes per ovary were less (P < 0.05) from ovaries bearing a corpus luteum (CL). Techniques used for oocyte recovery included slicing, follicle puncture and aspiration. The oocyte recovery rate was greatest (P < 0.05) using slicing. The average number of visible surface follicles was 5.20 +/- 0.97 with mean numbers of 2.5, 1.2, 0.82 and 0.62 per ovary for follicles sized 4, 8, 12 and 12mm respectively. Histological studies revealed large numbers of primordial follicles in prepubertal and atretic follicles in senile buffaloes. They also established a biphasic relationship of growth between oocyte diameter and follicular size.     

Histomorphological and histochemical studies on the female genitalia of aging goat. I. Lipid histochemistry of ovary

Paraffin sections of ovaries from 24 goats stained by Acetone Sudan Black B method for bound lipids and Copper-Phthalocyanin method for phospholipids revealed at least two categories of interstitial cells containing sudanophilic lipids with phospholipids. First category were those which were assumed to be formed from theca interna cells of atretic follicles. Second category included those originating probably from the granulosa cells of primordial follicles and form a continuous zone under the tunica albuginea. Another zone of interstitial gland cells presumed to be originated from the germinal epithelium has also been recognized under the latter. Appearance of sudanophilic material in relation to atresia of follicles in goat ovary has been reported.     

Blood pressure control programs on and off the worksite

Cows on high and low feeding levels were submitted to palpation of the ovaries and recording of the presence and size of follicles at various days of the cycle. A mid-cycle rise in follicular activity was apparent and for which the nonovulating ovary was responsible. High level feeding stimulated ovarian activity in each of the 2 ovaries throughout the cycle and follicular activity commenced earlier in the ovulating ovary on high level feeding than on low level feeding. A larger number of inseminations per conception was required by cows on high level feeding on low level feeding. The effect of ovarian activity on fertility is discussed.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

The role of three-dimensional ultrasonographic images in ovarian measurement

OBJECTIVE: To determine whether three-dimensional ultrasonography can increase the ability to evaluate the ovary and follicles, especially in women with polycystic ovary disease (PCOD). DESIGN: Prospective clinical study. SETTING: University hospital endocrine and infertility center. PATIENT(S): Sixty-six patients undergoing routine gynecologic evaluation were divided into two groups: the normal control group consisted of 22 patients, and there were 44 patients with PCOD. INTERVENTION(S): Three-dimensional ultrasonography was performed to store and document whole volumes of the ovaries for evaluation. MAIN OUTCOME MEASURE(S): Three perpendicular planes of bilateral ovaries are rotatable to obtain the largest dimensions. The three-dimensional volume was measured using the trapezoid formula. RESULT(S): The ovaries of the patients with PCOD were larger in size, area, and volume than those of normal controls. CONCLUSION(S): Stroma and volume determinations can be obtained more accurately by three-dimensional images than by traditional ultrasonography. Three-dimensional ultrasonography not only facilitates noninvasive evaluation and volume calculation but also completes the examination in a short time without increasing patient discomfort.     

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.     

Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad

Oocyte loss, either directly through attrition (germ cell death) or indirectly through follicular atresia (somatic or granulosa cell death), is a fundamental event associated with defining the time of normal or premature reproductive senescence in females. Although apoptosis has been reported to function as the underlying mechanism responsible for death of both germ cells and somatic cells in the ovary, the final molecular steps which commit ovarian cells to death have not been fully elucidated. To examine if death repressor activity of the bcl-2 gene product is important for germ cell survival, we conducted studies using a Bcl-2 loss-of-function (bcl-2 -/-) transgenic mouse model. Histological analyses revealed that ovaries collected from bcl-2 -/- mice possessed numerous aberrantly formed primordial follicle-like structures containing a single layer of granulosa cells without an oocyte. Additionally, the total number of primordial follicles present which contained a healthy oocyte was markedly reduced in bcl-2 -/- mice as compared to heterozygote (bcl-2 -/+) or wild-type (bcl-2 +/+) mice, suggesting that expression of the bcl-2 death repressor gene is critical for endowment of a normal complement of germ cells and primordial follicles in the mammalian ovary.     

Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization

OBJECTIVE: To assess the value of heparinized saline as a flushing medium for oocyte recovery. DESIGN: Prospective randomized study. SETTING: Academic tertiary referral center for fertility treatment. PATIENT(S): Thirty-five patients, with both ovaries intact having IVF-ET. INTERVENTION(S): Patients were randomized either to have the follicles of the left or right ovary flushed with heparinized normal saline at the time of oocyte recovery for IVF-ET. The contralateral ovary was flushed with heparinized culture medium. Oocytes obtained from each side were cultured separately and assessed for fertilization 18-21 hours after insemination. MAIN OUTCOME MEASURE(S): Collection and fertilization rates. RESULT(S): A total of 481 follicles were aspirated yielding 366 oocytes. Of these, 240 fertilized. From the side flushed with saline 185 oocytes were collected from 237 follicles, which was not significantly different from 181 oocytes collected from 244 follicles on the side flushed with culture medium (odds ratio = 1.23; 95% confidence interval = 0.79-1.92). Similarly, there was no significant difference observed in fertilization rates between oocytes obtained after saline (median 71.4%) and culture medium flush (median 75.0%) (odds ratio = 1.08; 95% confidence interval = 0.68-1.72). CONCLUSION(S): Heparinized normal saline is an equally good but cheaper and more convenient medium than standard heparinized culture medium and could replace it for flushing follicles during oocyte recovery for IVF-ET procedures.