Conditions for establishment of reflex ovulation in light estrous rats.

Continual anovulatory state associating with persistent vaginal cornification (light estrus) was induced by placing 4-day cycling rats under continuous lighting (LL). Uterine cervical stimulation was applied at arbitrary solar hours to light estrous rats showing continual vaginal estrus for more than 2 weeks. The ovulation was induced between 14 and 16 hr after the stimulation dissociating entirely with solar hours. Injection of anti-LHRH serum 5 min after the stimulation but not later than 20 min blocked this ovulation. Ovulation thus induced was always followed by pseudopregnancy with continual leucocytic vaginal smear lasting 10.70 days. The change in concentrations of peripheral serum progesterone during this period was almost similar to that of normal pseudopregnancy except extremely low levels observed at the start and end. Effectiveness of the cervical stimulation for induction of ovulation in light estrous rats was related to not only the duration of light estrus but also the time after transfer to LL, suggesting that the neural mechanism of ovulation in light estrous rats shifted from that of the spontaneous to reflex ovulators due to the extinction of environmental photic cue.     

Induction of ovulation and multiple ovulation in seasonally-anovulatory mares with equine pituitary fractions

Equine pituitary fractions were used to induce ovulation in seasonally-anovulatory pony mares. Over three experiments, 87% of mares ovulated following twice daily injections for 14 days of equine pituitary fractions. Of the mares which ovulated, 58% had 2 or more ovulations/estrus.     

Observation of ovulation in Macaca fascicularis

Objectives of the present study were to view the moment of ovulation, determine the postovulatory relationships between the ovary and egg mass, and to characterize ovulation in consecutive cycles of the Macaca fascicularis. Laparoscopic observation of the ovaries of 17 adult regularly cycling Macaca fasicularis was made during their menstrual cycles at the optimal time for detecting follicular development. Preovulatory morphology, follicular rupture and immediate postovulatory morphology were noted and photographed. Data are presented correlating the duration of the follicular phase and the luteal phase with that of the total cycle.     

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.     

Conditional induction of ovulation in mice

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.     

Control of ovulation in farm animals

Examination of hormonal changes occurring in farm species at the onset of puberty, during the follicular phase of the oestrous cycle, and at those times when ovarian activity is re-established after periods of seasonal or lactational anoestrus, provides circumstantial evidence that the final phases of follicular development are dependent on a pattern of tonic (episodic) LH secretion. A suppression of episodic LH secretion is associated with periods of anovulation. Stimulation of tonic LH secretion by repeated injections of small doses of synthetic Gn-RH or purified LH restores normal reproductive function in all but deeply anoestrous animals. Continuous infusion of Gn-RH is as effective as repeated injections. It is suggested that an additional inadequacy, possibly endocrine, contributes to the anovulatory state in deep anoestrus.     

Connective tissue breakdown in ovulation

A meshwork of collagen over the apical region of the follicle must be breached to permit the ovum to escape. We propose that specific collagenase activity is responsible for collagen breakdown in this region. Immature rats are primed with pregnant mare serum gonadotropin (PMSG), followed at 48 h by hCG. At 8 h after hCG, collagenase activity, measured in extracts of ovarian tissue, is elevated about five-fold. Ovulation follows at 10-12 h. Ovaries from PMSG-primed rats are dissected at 48 h, placed in a perfusion apparatus, and perfused with luteinizing hormone and 3-isobutyl-1-methyl xanthine. The ovulations induced by this treatment can be blocked to the extent of 70% with a synthetic collagenase inhibitor. The activation of procollagenase is believed to involve plasminogen activator and plasmin. In support of this, we find that tranexamic acid at 1 mM inhibits ovulation about 70%. The inhibitor must be added within 3-4 h of LH to be effective. A specific plasmin inhibitor, D-Val-Phe-Lys-chloromethyl ketone, is similarly effective.     

Mechanism of mammalian ovulation

The sequence of events within the ovary during the process of ovulation discussed in this review is schematically represented in Fig. 1. It is obvious that LH, perhaps with some contribution from FSH, is the normal physiological trigger for the ovulatory sequence of events, and it appears from the available information that the effects of LH are mainly mediated via adenylate cyclase and increased cAMP levels. The cAMP in turn, via cAMP-dependent protein kinase, influences at least three distinct steps in the ovulatory process which seem to be of crucial importance, namely 1) the stimulation of steroidogenesis; 2) the stimulation of cyclooxygenase/lipooxygenase leading to increased prostaglandin/leukotriene synthesis; and 3) the stimulation of plasminogen activator which catalyzes the conversion of plasminogen to plasmin. A fourth crucial step in the ovulatory mechanism is the LH-induced increase in latent collagenase, but it remains to be determined if this step is mediated via cAMP. Concomitant with the increase in latent collagenase, there also appears to be an LH-dependent increase in collagenase inhibitors. The latent collagenase is then activated, and it appears that leukotrienes and prostaglandins, as well as plasmin, may be involved in this process. The active collagenase causes a digestion of the collagen in the follicle wall, and plasmin, as well as possibly other proteolytic enzymes such as proteoglycanases, may cause a further dissociation of the follicular wall. These processes of digestion of collagen and dissociation of the collagen fibers result in an opening in the follicular wall with the formation of the stigma and rupture. While the weakening of the follicular wall takes place throughout the entire wall, rupture remains for the most part a localized process at the apex of the follicle. This localization of the rupture may be explained on the basis of mechanical factors operating when the follicle wall thins and weakens. While it is clear that prostaglandins and leukotrienes can influence smooth muscle by causing contractions and that these compounds can cause vascular changes such as increased permeability, vasodilation, and vasoconstriction, it is not clear what the exact role of these latter processes are in ovulation. It appears that progesterone and not estrogen play an important role in the mechanism of LH-induced follicular rupture, but the locus of action of progesterone and its mechanism of action remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuroendocrine control of ovulation

Modern methods of diagnosis have made the distinction between hypothalamic failure and ovarian failure routine. Failure of the orderly progression of hypothalamic gonadotrophin-releasing hormone (GnRH) → pituitary gonadotrophins → ovarian steroids and inhibin → hypothalamus/pituitary results in anovulation/amenorrhea. The hypothalamic connections that regulate the pattern and amplitude of GnRH pulses are plastic and respond to external/psychological conditions and internal/metabolic factors that may affect the hypothalamic substrate on which estrogen levels can act. We trace the neuroendocrine regulation of the ovarian cycle, concentrating on hypothalamic connections that underlie negative and positive feedback control of GnRH and the complementary role of the adenohypophysis. The main hormone regulating this "central axis" and the development of the endometrium is estradiol which is exported from the developing ovarian follicles and thereby closes the feedback loop with follicle development. Progesterone and inhibin are also involved. Neuroendocrine responses to internal and external factors can cause anovulation and amenorrhea. Generally, these are accompanied by abnormal negative feedback between estradiol and the gonadotrophins; coexistence of low estradiol and luteinizing hormone/follicle-stimulating hormone. There are three main causes: (1) genetic diseases that interfere with the migration of GnRH cells into the brain or result in misfolding of GnRH; (2) input from the brain that interrupts normal feedback (e.g. stress and weight loss amenorrhea); and (3) the effect of agents which alter central neurotransmission and hypothalamic function (e.g. elevated prolactin and psychotropic medications). All types of hypothalamic insufficiency result in insufficient stimulation of the ovaries. In addition to amenorrhea, this central alteration also results in other complications (downstream disease) that make hypothalamic amenorrhea of greater consequence than simply reproductive failure. Thus, there may be more at stake in the diagnosis and treatment of hypothalamic failure than brings the patient to her caregiver.     

The role of plasminogen activator in ovulation

The role of plasminogen activator in ovulation was investigated using the inhibitor, trans-aminomethylcyclohexane carboxylic acid (t-AMCHA). In the regular cycle rat, the plasminogen activator activity of the follicles increased from the diestrus to the estrus phase. In the latter phase, a proteolytic enzyme which was not inhibited by t-AMCHA appeared. After ovulation, the plasminogen activator activity decreased. When ovulation was induced in immature rats by pregnant mare serum gonadotrophin and human chorionic gonadotrophin, remarkable fibrinolytic activity appeared in the ovaries immediately before ovulation. When t-AMCHA was given in the ovulation-induced rats, the fibrinolytic activity of the ovaries was suppressed, the number of ovulated ova decreased and the timing of ovulation was delayed. When t-AMCHA solution was given to rats in the proestrus phase, ovulation was almost completely suppressed, but aprotinin solution exerted no effect on ovulation. These results suggest that plasminogen activator is a key enzyme in ovulation, and that the chain reaction from plasminogen activator to proteolytic enzyme (including collagenase) is of greater importance than that of plasminogen activator to plasmin.