Atretic Preantral Follicles Research Articles

Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00h. Animals were sacrificed 24h after surgery. Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. At 11:00h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Morphology and morphometry of preantral follicles, and immunolocalization of angiogenic factors in ovarian tissue from the neotropical primate Sapajus apella.

SummaryOvarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles from Sapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

Sensory denervation with capsaicin reduces ovarian follicular development and delays the onset of puberty in guinea pigs

Introduction: It has been documented that mammalian ovaries receive sympathetic, parasympathetic and sensory nerve fibers. The aim of this work was to investigate the effects of sensory denervation with capsaicin at the first vaginal opening (FVO) on follicular development and the expression of TRPV1 receptors in ovary cells as well as in the dorsal root ganglia (DRGs) and lumbar dorsal spinal cord neurons of guinea pigs. The DRGs and lumbar dorsal spinal cord neurons serve as a nerve connection from the ovaries to the CNS. Materials and Methods: Female guinea pigs received a subcutaneous injection of capsaicin (30 mM) at 10 days of age (P10), while control animals were injected with vehicle. Using light microscopy, we counted healthy preantral follicles (HPF), healthy antral follicles (HAF), atretic preantral follicles (APF), and atretic antral follicles (AAF) in the ovaries at the FVO, and the numbers of TRPV1-positive cells were counted in the ovarian follicles, DRGs, and lumbar dorsal spinal cord (L2-L4) neurons by immunohistochemistry. Results: Guinea pigs treated with capsaicin showed a significant delay of FVO in comparison with the control animals (36 vs. 44 days). In the ovaries, the number of preantral and antral follicles decreased significantly. Additionally, the number of TRPV1-positive thecainterstitial cells of the antral follicles was reduced significantly, and the number of TRPV1-positive neurons in the DRGs and lumbar dorsal spinal cord (L2-L4) decreased. Thus, we showed that TRPV1 receptors throughout the sensory fibers modulate ovarian follicular development and the onset of puberty in guinea pigs. Conclusion: Sensory denervation decreases ovarian follicular development and delays the onset of puberty of guinea pigs. Our data support the idea that through TRPV1 receptors, ovarian afferent fibers sense local stimuli that are sent to the CNS.

Somatostatin 14 affects the pituitary-ovarian axis in infant rats

The effects of multiple somatostatin (SRIH-14) treatment on the pituitary-ovarian axis were examined in infant rats. Female Wistar rats received subcutaneously two daily 20 μg/100g b.w. doses for five consecutive days (from 11 to 15 days of age). Changes in cell volume, volume density and number per unit area (mm²) of follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) immunolabeled cells were evaluated by stereology and morphometry. Serum FSH and LH concentrations were determined by RIA. Ovaries were analyzed by simple point counting of follicles. SRIH-14 treatment significantly reduced FSH and LH cell volume, while their volume density and number per unit area were unaltered. Serum concentrations of FSH and LH were significantly reduced. Volume and volume density of GH cells was significantly decreased after SRIH-14 treatment, while their number per unit area was unaltered. In the ovary, SRIH-14 induced a significant increase in the percentage of primordial follicles followed by a significant decrease in percentage of primary follicles. The number of healthy and atretic preantral follicles was unchanged. It can be concluded that SRIH-14 treatment during the infantile period markedly inhibits pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acts by inhibiting initial folliculogenesis without affecting atretic processes.

132. ACTIVIN A HAS A STIMULATORY EFFECT IN VITRO ON EARLY FOLLICLE DEVELOPMENT IN RAT OVARIES

Activins are dimers of inhibin β subunits and are growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily (1). Both βA and βB subunits are highly expressed in rat granulosa cells, while theca cells express little or no β subunit mRNAs (2). Oocytes lack expression of either subunit (3, 4). Activin is suggested to facilitate the responsiveness of granulosa cells to FSH (5). We hypothesized that activin, with or without FSH, could enhance the transition from the primordial to later preantral stages of follicle development. In two independent experiments, day 4 rat ovaries (n = 3 from different rats per treatment) were randomly assigned and cultured (6, 7) for 10 days in DMEM/Hams F-12 media with either no additives, FSH (100 ng/mL), activin A (50 ng/mL), or both. Day 4 fresh ovaries were also used as controls. Media and treatments were refreshed every alternate day. Ovaries were fixed andsectioned, or placed into Ultraspec for RNA extraction and real-time PCR analysis. Follicle numbers were counted as described previously (7). The proportion of atretic follicles (TUNEL staining) was determined in 3 randomly selected sections per ovary. Primordial follicles in all treatment groups were approximately 20% of those in Day 4 fresh ovaries. Primary follicles increased significantly (P < 0.05) only in the combined treatment group, where preantral follicles increased significantly (P < 0.0001) only when treated with activin A alone. Activin A alone decreased the proportion of atretic follicles in the primary and preantral classes, where the combined treatment increased the proportion of atretic preantral follicles. Real-time analysis revealed that expression levels of follistatin, FSH receptor and activin βA and βB subunits were all expressed at significantly higher levels in the Activin A-only treated group (P < 0.05). In summary, there was no effect on primordial follicle activation by any treatment. Activin alone had a stimulatory effect in vitro on subsequent folliculogenesis, but in the presence of FSH its effect was counteracted shown by an increase in atresia. Reasons for an increase in atretic preantral follicles in the combined treatment group are unclear. These studies support a stimulatory role for activin A in early follicle development and confirm the in vivo effects of activin on folliculogenesis (4). NHMRC program grant # 494802 and Fellowship (# 441101) provided financial support. (1) Vale W et al. 1986. Nature 321: 776–779.(2) Meunier H et al. 1988. Proc Natl Acad Sci USA 85: 547–251.(3) Roberts V et al. 1993. Journal of Clinical Endocrinology & Metabolism 7: 1402–1410.(4) Sidis Y et al. 1998. Biology of Reproduction 59(4): 807–812.(5) Drummond A et al. 2002. Endocrinology 143 (4): 1423–1433.(6) Nilsson E et al. 2001. Molecular and Cellular Endocrinology 182 (2): 145–155.(7) Rosairo D et al. 2008. Reproduction 136: 799–809.

Immunohistochemical localization of CD31, NOTCH1 and JAGGED1 proteins in experimentally induced polycystic ovaries of immature rats

We analyzed histomorphometrical changes and blood vessel immunohistochemical staining of CD31, NOTCH1 and JAGGED1 in induced polycystic ovaries of immature female Wistar rats, as well as serum hormone levels. The rats were randomly divided into control (n=18) and treated (n=18) groups. Treated animals received intramuscularly testosteronenantat weekly (0.1mg/g). Controls received the same amount of ricinus oil. Rats were weighed daily. Control and treated subgroups (6 rats per subgroup) were subsequently sacrificed after 21, 28 and 35 days of treatment. In ovaries of treated rats we found large cystic follicles, thick stromal tissue, many atretic preantral follicles, no ovulation and a thinner granulosa cell layer. CD31 stained blood vessels in the theca layer were reduced, with reduced JAGGED1 and NOTCH1 immunostaining. In controls, preantral and antral follicles were larger than in the treated group. Treated animals showed statistically significant lower progesterone and higher testosterone levels. They gained more weight than controls. Reduced immunostaining for NOTCH1 and JAGGED1 of reduced blood vessels of the theca layer was found in all stages of folliculogenesis with a distinct reduction in cystic and atretic follicles. Our results provide evidence of intrinsic abnormality during all stages of folliculogenesis in polycystic ovaries and this may result from crosstalk between circulating gonadotropins and follicular angiogenesis.

Cell-type-specific expression of murine multifunctional galectin-3 and its association with follicular atresia/luteolysis in contrast to pro-apoptotic galectins-1 and -7

Galectin-3 is a multifunctional protein with modular design. A distinct expression profile was determined in various murine organs when set into relation to homodimeric galectins-1 and -7. Fittingly, the signature of putative transcription-factor-binding sites in the promoter region of the galectin-3 gene affords a toolbox for a complex combinatorial regulation, distinct from the respective sequence stretches in galectins-1 and -7. A striking example for cell-type specificity was the ovary, where these two lectins were confined to the surface epithelium. Immunohistochemically, galectin-3 was found in macrophages of the cortical interstitium between developing follicles and medullary interstitium, matching the distribution of the F4/80 antigen. With respect to atresia and luteolysis strong signals in granulosa cells of atretic preantral but not antral follicles and increasing positivity in corpora lutea upon regression coincided with DNA fragmentation. Labeled galectin-3 revealed lactose-inhibitable binding to granulosa cells. Also, slender processes of vital granulosa cells which extended into the zona pellucida were positive. This study demonstrates cell-type specificity and cycle-associated regulation for galectin-3 with increased presence in atretic preantral follicles and in late stages of luteolysis.

Identification and localization of deoxyribonuclease I in the rat ovary.

Our objective was to identify the endonuclease responsible for DNA degradation in the ovary and determine its localization relative to the developmental state of ovarian follicles. Immature rats were treated with diethylstilbestrol (DES; DES group), DES + eCG (eCG group) or DES + eCG + hCG (hCG group). Nuclei of the eCG and hCG but not the DES group contained a 32/34-kDA DNase I-like endonuclease activity that was Ca2+/Mg(2+)-dependent, stimulated by Mn2+, optimal at pH 7, and identified by anti-DNase I antibody. G-actin, Zn2+, dithiothreitol, aurintricarboxylic acid, and sodium aurothiomalate, but not iodoacetic acid, inhibited the activity. Addition of eCG nuclear protein extracts to nuclei from the DES group induced oligonucleosomal DNA fragmentation, which could be prevented by pretreatment of the extracts with anti-DNase I antibody. DNase I was immunolocalized in nuclei of healthy luteal cells, antral follicle granulosa cells, oocytes of preantral follicles, atretic preantral follicles, and testicular spermatogonia, but was not observed in granulosa cells of preantral follicles, theca cells, antral follicle oocytes, or testicular spermatocytes. Nuclear extracts of rat kidney, liver, and spleen, and bovine, chicken, and human ovaries displayed DNase I-like activity. These results suggest that an endonuclease indistinguishable from DNase I is responsible for ovarian apoptotic DNA degradation.

Regulation of IGFBP-4 and -5 expression in rat granulosa cells.

One of the central questions in reproductive physiology concerns how follicles in the ovary are selected during folliculogenesis. Although it has been well established that folliculogenesis is initiated and maintained by the gonadotropin, follicle-stimulating hormone (FSH), how follicle selection takes place in the ovary is still an enigma.1 Based on the notion that selection of the dominant follicle might be due to the action of a locally produced inhibitor in the follicular fluid which could block the effects of FSH on the non-selected follicles, our laboratory initiated a project in 1988 to isolate and identify the FSH inhibitor present in porcine ovarian follicular fluid. Using a well-defined rat granulosa cell culture assay to monitor the purification, a polypeptide was isolated which exhibited a potent inhibition of FSH-stimulated estradiol production in those cells.2 Chemical characterization of the inhibitor, however, revealed that its structure corresponded to insulin-like growth factor binding protein-3 (IGFBP-3).3 Subsequent studies carried out by our laboratory identified that the rat ovary produced five IGFBPs in a tissue-specific manner, with the mRNA for IGFBP-2 mainly expressed by theca interstitial cells, IGFBP-3 by luteinized granulosa cells, IGFBP-4 by the granulosa of atretic antral follicles, IGFBP-5 by the granulosa of atretic preantral follicles and IGFBP-6 by the theca externa, stroma and smooth muscle cells4–6. IGFBP-1 was not detected in the rat ovary. These findings indicate that the transcription of each IGFBP gene is regulated differently in the rat ovary and that each IGFBP may have a different physiological function. This hypothesis is strengthened by our finding that exogenously added IGFBP-2, -3, -4 and -5 were able to attenuate the FSH-stimulated production of estradiol and progesterone in cultured rat granulosa cells7,8 and ovarian intrabursal injection of IGFBP-3 was able to decrease the ovulation rate of pregnant mare’s serum gonadotropin/human choriogonadotropin-primed animals.9 KeywordsLuteinizing HormoneGranulosa CellFollicular FluidLuteinizing Hormone ReceptorLuteinized Granulosa CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Localization of insulin-like growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle.

To investigate the potential role of insulin-like growth factor-binding protein-5 (IGFBP-5) in ovarian physiology, we employed in situ hybridization and Northern analysis to localize IGFBP-5 mRNA in rat ovaries during the estrous cycle. By Northern analysis, the mRNA was abundant at all stages of the cycle. Two species of mRNAs were detected, with sizes of 6.0 and 1.8 kilobases, respectively. The relative amounts of the two transcripts changed throughout the cycle. By in situ hybridization, IGFBP-5 mRNA was expressed in only a few cell types: 1) granulosa cells of some atretic follicles, 2) some secondary interstitial cells, 3) some corpora lutea, and 4) the surface epithelium. The levels of message in both the granulosa and secondary interstitial cells changed over the cycle. At 1000 h on proestrus (before the LH/FSH surge), the message was expressed in only a few follicles. Interestingly, all were small atretic preantral (200-250 microns) follicles. At 2000 h on proestrus (after the LH/FSH surge), the IGFBP-5 mRNA was more abundant; now almost every atretic preantral follicle showed a strong hybridization signal. At 0200 and 1000 h on estrus, the mRNA appeared for the first time in granulosa cells of some atretic antral follicles and in secondary interstitial cells. Hence, virtually all atretic follicles, preantral and antral, now showed IGFBP-5 gene expression. In contrast to that on proestrus and estrus, the hybridization signal on diestrous days 1 and 2 was much less prominent and was found in only a few atretic preantral follicles. Throughout the cycle, IGFBP-5 mRNA was evident in some corpora lutea, but it was not particularly prominent. Abundant IGFBP-5 mRNA was evident in the surface epithelium, and no change was detected over the cycle. Dominant follicles were devoid of IGFBP-5 mRNA. In conclusion, this paper presents the first evidence that the IGFBP-5 gene is expressed in the adult rat ovary. The IGFBP gene is expressed in a cell-specific manner, e.g. in atretic granulosa, secondary interstitial cells, corpora lutea, and the surface epithelium, and the stage of the cycle significantly affected message levels, especially in atretic granulosa and secondary interstitial cells around estrous morning. These findings suggest that IGBP-5 may be an autocrine/paracrine regulator of ovarian physiology, particularly in relation to preantral follicle atresia.

Gonadotropin-releasing hormone induces classical meiotic maturation in subpopulations of atretic preantral follicles.

GnRH has been shown to induce premature meiotic maturation in preantral follicles of the immature estrogen-primed hypophysectomized rat. As these animals are free of circulating gonadotropins and contain large numbers of full-grown oocytes in preantral follicles, we have investigated this model to determine its usefulness in studying meiotic maturation. We show that a maximum dose of the agonist D-Trp6,Pro9,Net-LRF (GnRH-a) induces approximately 25% of full grown oocytes to resume meiosis within a 12-h period. This response is dose dependent (ED50 = 0.24 microgram/rat) and specific for GnRH-a. GnRH-a stimulates germinal vesicle breakdown and first polar body formation within 2 and 8 h, respectively. More than 75% of those oocytes that initiate meiotic maturation reach metaphase II by 15 h. This effect of GnRH parallels the time course of physiological meiotic maturation triggered by LH as well as that of oocytes maturing spontaneously in vitro. Oocytes in primordial and primary follicles do not respond to GnRH. The majority of affected follicles are small tertiary follicles (200-400 micron in diameter) and show signs of atresia. This atresia is not caused by GnRH-a and does not, in itself, result in meiotic maturation, but appears to confer susceptibility to GnRH-a-induced meiotic maturation. Our studies indicate that this animal model will be useful to elucidate further the mechanisms and requirements for meiotic maturation. It will also facilitate investigation of the role of atresia in the GnRH response of tertiary follicles and the issue of follicle heterogeneity within these animals.

Aging and ovarian function in the white-footed mouse (Peromyscus leucopus) with specific reference to the development of preovulatory follicles.

A study was made of the effects of increasing age on uterine histology, follicular development and steroidogenesis within the ovary of the white-footed mouse (Peromyscus leucopus). The animals were autopsied on each day of the estrous cycle and ranged from 14 to 49 mos. of age. The data indicated that the animals maintained estrous cycles throughout their lifespan as judged by cyclic changes in uterine histology. In addition these studies showed that aging (1) did not alter ovarian concentrations of testosterone, 17 beta-estradiol or progesterone, (2) resulted in a decrease in the number of primary and preantral follicles during metestrus, proestrus and estrus, (3) increased the percentage of atretic preantral follicles during metestrus only, and (4) did not reduce the number of antral (preovulatory) follicles that develop by proestrus. These observations suggest that in P. leucopus the "rescue" of preantral follicles constitutes the mechanism which compensates for the decrease in the number of smaller follicles and allows the normal number of preovulatory follicles to develop and ovulate. It is also possible that this mechanism exists in the laboratory mouse and rat since species-specific numbers of preovulatory follicles develop in aged cycling animals despite an age-related decrease in the total follicular populations.