Estradiol esterification in the human preovulatory follicle.

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K(m) and V(max) values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.     

Differences in preovulatory follicle dynamics and timing of preovulatory LH surge affect fertility of maiden sheep reared in semi-arid extensive conditions

In the current study follicular dynamics, pituitary function, ovulatory response and luteal activity of 30 maiden Barbarine sheep were analyzed according to oestrus occurrence and lambing outcome after oestrus synchronisation with cloprostenol. Animals were retrospectively classified in three groups named as O? (n = 7, ewes not displaying oestrus), O+L? (n = 7, ewes showing oestrus but failing to lamb) and O+L+ (n = 16; ewes showing oestrus and lambing thereafter). All the sheep ovulated and daily transrectal ultrasonographies revealed that preovulatory follicles were present at cloprostenol injection in all the animals. In sheep O+L+ and O+L?, 50% and 57% of the ovulatory follicles were the largest follicles at cloprostenol treatment (mean size of 4.1 ± 0.26 mm and 4.3 ± 0.74 mm, respectively). In O? ewes, the same percentage was higher (86%, P < 0.05 when compared to group O+L+; mean size of 4.0 ± 0.46 mm). The number of large follicles and the final diameter of the ovulatory follicles at oestrous tended thereafter to be higher in group O+L+ (1.4 ± 0.1 and 6.4 ± 0.2) than in groups O+L? (1 ± 0.2 and 5.7 ± 0.36) and O? (0.9 ± 0.2 and 5.9 ± 0.5, respectively). Conversely, the number of medium follicles at oestrus detection was higher in the group O+L? (2.1 ± 0.3, P < 0.05) than in the other two groups (1 ± 0.2 and 1 ± 0.3 for O+L+ and O? respectively). Timing of preovulatory LH surge was earlier for ewes O? (24.0 ± 4.75, P < 0.05) than for sheep O+L+ and O+L? (37.9 ± 2.45 h and 38.0 ± 4.75 h, respectively) and 94% of O+L+ ewes had a LH surge between 16 h and 64 h after cloprostenol injection compared to 57% in O+L? and O? groups (P < 0.05). Thus, maiden Barbarine sheep failing to display oestrus or conceive showed alterations in their follicular dynamics and, thereafter, pituitary function and ovulatory response.     

Ovarian follicle growth and consequences for fertility in sheep

Understanding the pattern of ovarian follicle development is seen as an important step leading to the development of techniques that maximise fertility in sheep. Repeated observations of the growth of individual follicles have led to the understanding that follicles develop in a wave-like pattern during the oestrous cycle, with two to four waves per cycle being the most common. The ease with which follicle waves are described seems to depend on the their frequency and the number of follicles per wave. There is evidence for the largest follicle(s) of a follicle wave inhibiting the development of other follicles; however, in some cases this is not apparent as other follicle waves emerge when a previous large, healthy follicle is still present. Follicle development can be manipulated using exogenous gonadotrophins or progestagens and these have been shown to alter the number or age profile of developing follicles. The ovulation of aged follicles in cattle clearly has a detrimental effect on fertility, but this relationship is less clear and seems to be less critical in sheep. Recent findings at the molecular level show that the bone morphogenetic proteins (BMP) and their receptors are critically involved in the control of ovulation rate, but fully understanding their mechanism remains to be described. This highlights the potential for the integration of molecular and physiological findings to better develop methods to manipulate follicle development and reproduction in sheep.     

Ultrasonic evaluation of the preovulatory follicle in the mare

Ultrasonically visible characteristics of preovulatory follicles in mares which single ovulated were studied daily for 79 preovulatory periods in 40 mares. The preovulatory follicle became the largest follicle in the ovary from which ovulation later occurred six or more days before ovulation in 65 of 79 (82%) preovulatory periods; the mean was day -7 (range, day -14 to day -4). The increase in mean diameter of the preovulatory follicle was linear (R(2)=99.5%) over day -7 (29.4 +/- 0.8 mm) to day -1 (45.2 +/- 0.5 mm; growth rate, 2.7 mm/day). Follicles which double-ovulated were smaller (P<0.05) on day -1 (36 +/- 1.6 mm; n=12 follicles). Preovulatory follicles exhibited a pronounced change in shape from a spherical to a conical or pear-shaped structure in 84% of the preovulatory periods. Remaining follicles retained a spherical shape. Scores representing thickness of the follicular wall increased (P<0.05) as the interval to ovulation decreased. There was no significant difference among days in mean gray-scale value of the follicular wall or in echogenicity of the follicular fluid. Although diameter and shape of the follicle and thickness of the follicular wall changed during the preovulatory period, no reliable ultrasonically visible predictor of impending ovulation was found.     

Distribution of binding sites for human chorionic gonadotropin in the preovulatory follicle of the rat

The distribution of binding sites for human chorionic gonadotropin (hCG) in the preovulatory follicle was studied by autoradiography. An ovulatory dose (10 IU/rat) of [125I]hCG (1.4 muCi/IU) was administered intravenously, and large Graafian follicles were isolated 3 h later by microdissection. Injection of excess unlabeled hCG (500 IU/rat) prevented uptake of radioactivity by the follicle, indicating that binding of iodinated hormone was confined to specific and saturable receptor sites. The density of bound hormone molecules was highest in the theca interna and in three to four layers of mural granulosa cells adjacent to the basement membrane; labeling was chiefly associated with the cell borders. No significant binding could be detected either on the oocyte or on the cumulus cells surrounding the oocyte. We therefore suggest that the induction of ovum maturation does not require attachment of the hormone to the oocyte itself or to follicle cells in its immediate vicinity.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

Specialised plasma membranes in the preovulatory follicle op the fowl

Summary In the hen's follicle processes from the zona granulosa cells extend into the zona radiata. The terminal plasma membrane of these processes has a total thickness of around 160 Å and consists of five layers. Small granules spaced at regular intervals are attached to the cytoplasmic aspect of the inner layer by short stalks. In the 2 mm and 7 mm follicles the plasma membrane of the ovum facing the specialised terminal membranes has a striated appearance and shows a regular arrangement of granules attached by stalks to both its inner and outer aspects. The terminal and striated membranes are separated by an interval although there are areas of closer contact. In the 15 mm and 35 mm pre-ovulatory follicles the plasma membrane round the whole surface of the ovum is now typical striated membrane with bristles and attached granules. No explanation can be given at present of the function of the terminal membranes of the granulosa processes. They may indicate some change in the permeability permitting the intercellular diffusion of particles. It is suggested that the striated ovum plasma membrane is associated with the adsorption and transport of substances into the ovum for yolk synthesis.     

Ultrasonic characteristics of preovulatory follicle and ovulation in Caspian mares

The Caspian breed of horses is believed to be the direct descendant of the earliest equine animals. Some special characteristics of Caspian horse differentiate this breed of horses from other breeds. In the current study the ultrasonically observed characteristics of a preovulatory dominant follicle and the lengths of estrus, diestrus as well as some related parameters were studied during 42 interovulatory intervals in 11 healthy Caspian mares. The preovulatory dominant follicle deviated from subordinate follicles and became the largest follicle in the ovaries at Day −8.7±0.53 (Day 0=ovulation). Every mare was a single ovulator with ovulations more frequent from the left ovary than from the right (65% versus 35%). Mean length of estrus, diestrus, and interovulatory interval were 8.3±0.86, 13.8±0.59, and 22.1±0.40 days, respectively. The time interval from ovulation until the time in which the mares were no longer in estrus was 1.9±0.42 days.     

mTOR controls ovarian follicle growth by regulating granulosa cell proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.     

Antral follicle growth and endocrine changes in prepubertal cattle,sheep and goats

In the growing heifer calve, there is an early post-natal, gonadotrophin driven increase in ovarian antral follicle growth. The endocrine regulation of and reason for this initial stimulation of ovarian follicular development are not fully understood. This initial endocrine activity appears to be later held in check by negative feedback suppression mechanisms until the heifer is of a sufficient body size to initiate oestrous cycles and to reproduce. There is increasing evidence from recent ultrasonographic studies, performed in the same groups of prepubertal heifer calves, that the development of ovarian antral follicles and tubular genitalia occur in parallel. There appear to be two distinct periods of enhanced development of the reproductive organs, from 2 to 14 weeks of age and again from 34 to 60 weeks of age, or just prior to puberty. First ovulation in heifers is preceded by a gradual increase in pulsed LH secretion, which results in enhanced antral follicle development and oestrogen production. It was demonstrated that prepubertal heifers produced recurrent antral follicular waves; maximum sizes and life span of the dominant follicles of waves, as well as periodicity and FSH dependency of wave emergence were similar to those in adult cattle. In does, no Graafian follicles are seen at birth and total follicle numbers increase to 2 months of age, and then decline to 5 months of age. In ewe lambs, studies using transrectal ovarian ultrasonography showed that antral follicle recruitment and growth increased after the first 2 months of age and just before puberty. This bi-phasic pattern of changes in ovarian follicle recruitment and growth is strikingly similar to that in heifer calves, but it contrasts with earlier post-mortem examinations of ovaries in ewe lambs. Unlike in cattle and adult ewes, the rhythmic pattern of follicular wave emergence was not established in pre- and peripubertal ewe lambs. The early increase in antral follicle numbers and size in ewe lambs may be, at least in part, due to changes in FSH release and potency, and enhanced follicle production prior to first ovulation is probably caused by an increase in the frequency of LH pulses.     

Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.     

Factors regulating endogenous retroviral sequences in human and mouse

Endogenous retroviruses (ERVs) are stably integrated in the genome of vertebrates and inherited as Mendelian genes. The several human ERV (HERV) families and related elements represent up to 5-8% of the DNA of our species. ERVs may be involved in the regulation of adjacent genomic loci, especially promoting the tissue-specific expression of genes; some HERVs may have functional roles, e.g., coding for the placental fusogenic protein, syncytin. This paper reviews the growing evidence about factors that may modulate ERVs, including: cell and tissue types (with special attention to placenta and germ cells), processes related to differentiation and aging, cytokines, agents that disrupt cell functions (e.g., DNA hypomethylating agents) and steroids. Special attention is given to HERVs, due to their possible involvement in autoimmunity and reproduction, as well as altered expression in some cancer types; moreover, different HERV families may deserve specific attention, due to remarkable differences concerning, e.g., expression in tissues. A comparison with factors interacting with murine ERV-related sequences indicates that the mouse may be a useful model for studying some patterns of HERV regulation. Overall, the available evidence identifies the diverse, potential interactions with endogenous or exogenous factors as a promising field for investigating the roles of ERVs in physiology and disease.     

Relationship between follicle growth and circulating gonadotrophin levels during postnatal development of sheep

This study investigates the number and size of ovarian antral follicles in relation to plasma follicle stimulating hormones (FSH) and luteinizing hormone (LH) concentrations from birth to 26 weeks of age in ewe lambs of the Ouled Djellel breed, a non-seasonal breed of sheep. Plasma was collected from 10 ewe lambs at 14 sampling times (Week 0, i.e. <24h, Week 1 and every two weeks from Week 4 to Week 26, inclusive). At each of these stages, four ewe lambs were slaughtered, the ovaries recovered and weighed, and the number and size of the follicles determined from histological examination. The pattern for plasma FSH showed a peak at Week 10, a smaller peak at Week 18 and a very small peak at Week 24. The pattern for LH was similar until Week 24 when the largest peak occurred. Paired ovarian weight increased rapidly from birth to four weeks and then more slowly to 10 weeks, followed by a decline at 12 weeks and a gradual increase from 14 to 24 weeks of age. The number and total diameter of follicles > or =3 mm in diameter showed similar patterns of development--rising gradually from birth to Week 14, falling to Week 16 and then rising more rapidly to a peak at Week 24. Maximum follicle diameter declined from birth to Week 1, then rose rapidly to Week 4, followed by a more gradual rise to Week 14 and, thereafter, a more rapid increase to a peak of 7.23+/-0.16 mm at 24 weeks old. The number of follicles (<3 mm diameter) increased rapidly from birth to Week 10 and then declined to values similar to those at Weeks 1 and 4. First behavioural oestrus was observed at Week 24 and a corpus luteum was present on the ovary of one lamb at Week 24 and two lambs at Week 26. It was concluded that two or three peaks in plasma FSH and LH levels precede puberty and first ovulation in Ouled Djellel ewe lambs, and first ovulation occurred at 24-26 weeks of age. The increase in follicle number and size generally reflected the pattern of plasma FSH and LH levels.     

Progesterone exposure of the preovulatory follicle in the seasonally anestrous ewe alters the expression of angiogenic growth factors in the early corpus luteum

Gonadotrophin releasing hormone (GnRH)-induced ovulation in seasonally anestrous ewes is associated with a high incidence of defective corpora lutea (CL), which can be completely eliminated by priming ewes with progesterone before GnRH treatment, but the physiological basis of this has remained elusive. This study tested the hypothesis that progesterone priming eliminates defective luteal function by altering the expression of Vascular Endothelial Growth Factor (VEGF), its receptor VEGFR-2, and angiopoietin (ANG)-1, ANG-2 and their receptorTIE-2 in the early CL. Fifteen seasonally anestrous ewes were treated by i.m. injection with 20 mg of progesterone 3 days before the start of GnRH treatment, while another 15 animals served as controls. Intravenous injections of 500 ng GnRH were given to all the ewes every 2 h for 28 h, followed by a 300 μg GnRH bolus injection to synchronize the preovulatory luteinizing hormone (LH) surge. Corpora lutea were collected 1, 2 and 4 days after ovulation and analyzed for protein and mRNA expression of VEGF, VEGFR-2, ANG-1, ANG-2 and Tie-2 using Western Immunoblotting and in situ hybridization. VEGF, VEGFR-2 and ANG-1 expression was significantly higher (P ≤ 0.05) in the CL of progesterone-primed animals compared to non-primed ones. However, no differences were observed in the ANG-2 or Tie-2 expression levels between the two treatment groups. These data suggest that progesterone priming of the preovulatory follicle alters the expression of some angiogenic growth factors in the early CL, leading to greater vascular stability and thereby normal luteal function.     

Injection of soluble vascular endothelial growth factor receptor 1 into the preovulatory follicle disrupts ovulation and subsequent luteal function in rhesus monkeys

Remarkable changes in vascular permeability and neovascularization occur within the ovulatory, luteinizing follicle. To evaluate the importance of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in periovulatory events, sequential experiments were designed in which vehicle (PBS/0.1% BSA; controls, n = 13) or a low dose (1.5 micro g; n = 4) or a high dose (7.5 micro g; n = 4) of a VEGF antagonist, soluble VEGF receptor 1 (sVEGFR1) chimera, was injected directly into the preovulatory follicle of rhesus monkeys the day before (Day -1) or the day of (Day 0) the midcycle LH surge during spontaneous menstrual cycles. After vehicle injection, animals typically exhibited patterns and levels of serum progesterone (P(4)) that were comparable to those of untreated animals in our colony. Following low-dose sVEGFR1 injection, serum P(4) levels were diminished in two of four animals from the early to midluteal phase, but were similar to vehicle controls thereafter. In contrast, high-dose sVEGFR1 injection decreased serum P(4) levels throughout the luteal phase compared with levels in controls (P < 0.05), but it did not cause premature menstruation. Control follicles displayed indices of rupture (protruding stigmata) and luteinization. However, sVEGFR1-injected follicles exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization, but not necessarily timely ovulation. Histological evaluation of serial sections from ovaries removed on Day 3 after treatment revealed that all (n = 3) vehicle-injected follicles ovulated, whereas half (n = 3 of 6) the sVEGFR1-injected follicles failed to ovulate and still contained an oocyte in the antrum. No appreciable differences were apparent between treatment groups in numbers of cells in luteal tissue (Day 3 or 6 after treatment) that stained positive for immunochemical or histochemical markers of proliferative (Ki67), endothelial (platelet endothelial cell adhesion molecule 1), and steroidogenic (3beta-hydroxysteroid dehydrogenase) cells. However, there was a dose-dependent increase (P < 0.05) in extracellular space in the corpus luteum by midluteal phase in sVEGFR1-treated animals. The data suggest that acute exposure to a VEGF antagonist can impair ovulation, and the subsequent development and functional capacity of the primate corpus luteum. The results are consistent with a critical role for VEGF in normal ovarian function during the periovulatory interval in primates.