I. INTRODUCTION

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This review concerns the phenomenon of luteolysis or regression of the corpus luteum, which terminates the female reproductive cycle of many mammals. Luteolysis is characterized by an initial decline of progesterone secretion that is commonly designated as functional luteolysis as distinct from structural or morphological luteolysis which, as the name suggests, signifies the subsequent change in the cellular structure of the gland and its gradual involution in the ovary to form a small scar composed of connective tissue. This latter structure, known as the corpus albicans, persists in the ovary, often for several weeks. However, the distinction between functional and structural regression of the corpus luteum is not well defined and, indeed, they may constitute part of an ongoing process. Luteolysis appears to have evolved in some species as a mechanism to increase reproductive efficiency. In most regularly cycling mammals when the female does not conceive following ovulation, the corpus luteum which forms subsequently in the ovary is "removed" to permit a new ovarian cycle to begin. In this way, after a relatively short interval of time, a further opportunity is provided for the female to conceive. In a few mammals, typified by the dog, no mechanism appears to exist to curtail the life span of the corpus luteum and, in unmated females or those failing to conceive, the corpora lutea last for a period approaching the length of pregnancy. The main secretory product of the corpus luteum in most mammals is the steroid hormone progesterone, which is the hormone of pregnancy in mammals. Progesterone is considered to be essential for pregnancy maintenance by inducing a state of quiescence in the myometrium (128) and by suppressing the maternal immune response to fetal antigens (626, 627, 666). In addition to providing a uterine environment suitable for the development of the embryo, progesterone is also responsible for the reduction of cyclic ovarian activity during pregnancy in most mammals and is responsible, in part, for mammary development. The corpus luteum also plays a key role in regulating the length of the ovarian cycle in most cyclic mammals, and the extension of the life span of the corpus luteum and progesterone secretion is necessary in most species to maintain gestation in its early stages.

Although progesterone is the hormone which, from the evolutionary point of view, is responsible for viviparity, and in some cases ovoviviparity (98), this review discusses the regulation of the life span of the corpus luteum in cyclic mammals. Progesterone is also produced by the placenta in some species, and in these, the placenta usually becomes the dominant source of progesterone during the later stages of pregnancy, e.g., sheep, horse, and human. Such a change in the source of progesterone for pregnancy maintenance is designated as the luteoplacental shift. In other species such as the goat, pig, rabbit, and mouse, the corpus luteum (or in polyovulatory species, corpora lutea) remains the principal source of progesterone throughout pregnancy. In some of these species, such as the goat and mouse, the initiation of labor appears to involve a luteolytic mechanism. In species where the corpus luteum is the sole source of progesterone, ovariectomy (removal of the corpus luteum) will terminate pregnancy at any stage, whereas in those species where the placenta produces progesterone, the corpus luteum can be removed when placental production is high enough to maintain pregnancy. In women with ovarian dysgenesis, a donor fertilized ovum will develop in the uterus when exogenous progesterone and estrogen are given to replace the secretion by the corpus luteum. When placental steroid production is adequate to maintain pregnancy, pregnancy will continue to term without further administration of exogenous hormones (431, 512).

Coiter (117) described the presence of cavities filled with a yellow solid in the ovary, but it was de Graaf (146) who gave the first definitive description of these structures noting that their number appeared to be related to the number of fetuses in utero. Malpighi (440) provided an accurate microscopic description of these structures and was the first to apply the name corpus luteum, literally the yellow body. Beard (51) postulated that corpora lutea were responsible for the suppression of ovulation and estrus during pregnancy, and about that time, Prenant (566) suggested that the corpus luteum might be a gland of internal secretion directly benefiting the egg with which it appeared to be associated. It was, however, Fraenkel (218), a pupil of Gustav Born, who demonstrated that corpora lutea were necessary for implantation and the subsequent maintenance of pregnancy in the rabbit. Corner and Allen (124) and Allen and Corner (14) prepared a relatively pure alcoholic extract of corpora lutea from sows and showed that this extract maintained pregnancy in ovariectomized rabbits. A few years later, the isolation of the pure crystalline hormone was reported simultaneously by four groups (93, 290, 634, 731). Slotta et al. (634) named the compound progesterone and suggested a structural formula, and in the same year, the compound was synthesized by Butenandt and Westphal (92).

There is probably no area of mammalian physiology where interspecies variation is so manifest as in the endocrine regulation of the ovarian cycle. To appreciate the comparative aspects of luteolysis in controlling the reproductive cycle in mammals, a brief classification and description of the various types of cycles are outlined below. A more detailed description of reproductive cycles in mammals is described elsewhere (24, 622). Ovarian cycles of the more common eutherian mammals can be divided into five groups as follows: 1) primate menstrual cycle, e.g., monkey and humans; 2) domestic animal cycles, e.g., sheep, cow, goat, pig, horse, and guinea pig; 3) laboratory rodent cycles, e.g., rat, mouse, and hamster; 4) reflex ovulators, e.g., rabbit, cat, mink, ferret, and camel; and 5) canine cycle, e.g., dog and wolf.

As shown in Figure 1, the phenomenon of luteolysis occurs in all species in groups 1 and 2, whereas in groups 3 and 4, luteolysis occurs in some species primarily as a means of terminating pseudopregnancy. In group 5, there is no evidence for the occurrence of luteolysis so that the corpus luteum persists for a period equivalent to that of pregnancy.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Occurrence of luteolysis for controlling life span of corpus luteum during cycle or during pseudopregnancy in different species. E, estradiol-17; P, progesterone; LH, luteinizing hormone.

Canines exhibit only about two ovulatory cycles per year. Ovulation and corpus luteum formation occur under gonadotropic control. A functional corpus luteum is formed that secretes progesterone throughout pregnancy after a fertile mating. In unmated animals or after an infertile mating, the corpus luteum persists for a period approximating the normal duration of pregnancy. During pseudopregnancy, or so-called phantom pregnancy, females exhibit nest building and mammary development and often begin to lactate. Hysterectomy has no effect on the life span of corpora lutea in the dog, although it may shorten the duration of anestrus (316). It appears that a luteolytic mechanism has not evolved in these animals to curtail the life span of the corpus luteum when pregnancy fails to occur, and no signal from the embryo is required to extend the life span of the corpus luteum. Reproduction in these animals is rather inefficient, since they do not have repetitive cycles and thus they have an opportunity to conceive only about twice per year.

In marsupials such as the red kangaroo, "uterine" pregnancy is shorter than the length of the ovarian cycle, and the presence of an embryo in the marsupial pouch does not extend the cycle or inhibit subsequent ovulation (681). This is in contrast to mammals that show spontaneous cycles, ovulation, corpus luteum formation, and regression where the presence of an embryo in the uterus is required to extend the life span of the corpus luteum for varying periods of time during pregnancy. [For reviews on the effects of the embryo on corpus luteum life span, see Moor (499), Heap et al. (296), Webley and Hearn (708), Stouffer et al. (658), and Hamberger et al. (282); see also sect. VI]. It is likely that these differences in reproductive patterns are meaningful in terms of the adaption of species to reproduce more efficiently under different environments and life patterns. Comparative aspects of corpus luteum development and function have been reviewed previously (357, 524, 591).

Under the influence of the preovulatory surge of LH from the anterior pituitary, the mature follicle ruptures and expels the ovum. The wall of the follicle collapses in folds, and capillaries invade the developing corpus luteum probably under the influence of angiogenic and mitogenic factors that may include basis fibroblast growth factor (261, 517), platelet-derived growth factor (358), insulin-like factor I (661, 349), heparin binding growth factor (264), and vascular endothelial growth factor (572). In domestic animals and primates, the major luteotropin appears to be LH, a luteotropin being defined as a substance that promotes the growth of the corpus luteum and stimulates the production of progesterone. For example, hypophysectomy in primates (220, 746) and in sheep (351, 353) causes regression of the corpus luteum, an effect which can be reversed by exogenous LH. Also, immunoneutralization of circulating LH with antibodies against bovine LH causes regression of the corpus luteum in sheep (231, 455; see Fig. 2). In primates, progesterone secretion is dependent on the pulsatile secretion of LH throughout the luteal phase (220, 746), whereas in the sheep, secretion of progesterone appears to be independent of LH pulses, since secretion can be maintained with minimal basal levels of LH (478). It may be that pulsatile LH secretion is not an absolute requirement per se for corpus luteum function in primates, since GC is released in a continuous nonpulsatile fashion but clearly maintains the corpus luteum during the establishment of pregnancy (see sect. VI). Luteinizing hormone pulses appear to be necessary in the cow for development of a fully functional corpus luteum but, as in the sheep, only basal levels are required to maintain progesterone secretion later in the luteal phase (548). The reduction in progesterone during luteolysis removes the progesterone blockade of tonic gonadotropin releasing hormone (GnRH)/LH pulses, and the consequent increase in pulse frequency promotes the growth of the preovulatory follicle and increases estrogen secretion for the next cycle. Although pituitary prolactin released by a mating stimulus in rodents is critical for maintaining the corpus luteum in a progesterone-secreting mode (225), there is little evidence to support a role for prolactin in luteal function in ruminants or primates. For example, in sheep, the infusion of ovine prolactin into the ovarian artery did not stimulate progesterone secretion (473). Moreover, the administration of 2-bromo--ergocryptine, a potent inhibitor of prolactin secretion, did not affect progesterone levels or the cycle length (521). In primates, prolactin does not alter progesterone production by dispersed luteal cells from monkeys (656) or women (668) in the presence or absence of human CG (hCG). However, in the canine, both prolactin and LH are required to maintain the secretory function of the corpus luteum (121).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Progesterone secretion rate (solid bar) and ovarian blood flow () after systemic infusion of antiserum against bovine LH (3.84 ml/h for 12 h). Progesterone secretion rate showed a marked, but continuous, decline within 1 h of beginning infusion. [From McCracken et al. (455).]

In primates, the corpus luteum is a significant source of inhibin, a heterodimeric glycoprotein, which was initially found in the developing follicle and having an ascribed function of inhibiting follicle-stimulating hormone (FSH) secretion. Plasma levels of inhibin are elevated during the luteal phase in primates (476) and are reduced by GnRH antagonists and stimulated by hCG (640). It is possible that luteal inhibin in primates acts to block the release of pituitary FSH, thus accounting for the lack of follicular development during the luteal phase, whereas in species that do not produce large amounts of luteal inhibin, waves of follicular development occur during the luteal phase, e.g., sheep and cow. Different forms of inhibin appear to be secreted by the corpus luteum (inhibin A) versus the follicle (inhibin B) (269). Moreover, it has been shown that inhibin A will reduce bioactive FSH blood levels and prevent follicular development in monkeys (498). In primates, the "rescue" of the human corpus luteum with exogenous hCG results in increased inhibin production that may extend the inhibition of follicular development in early pregnancy (331). However, there is no evidence to suggest that inhibins are involved in the process of luteolysis in primates, at least in the marmoset monkey (223). Relaxin is also produced by the cyclic corpus luteum in several species including human (714), pig (620), and rat (258). However, the function of luteal relaxin is presently unclear, and no specific action of relaxin has so far been associated with luteolysis. Oxytocin is synthesized by the corpus luteum of some species, notably ruminants, and may play a contributory role in luteolysis (see sects. III and IV).

The overall rate of growth of the corpus luteum in most species is extremely rapid. For example, in the cow, the weight of the corpus luteum 3 days after ovulation averages 640 mg, whereas on day 14, the average weight is 5.1 g (201). Most of this rapid increase in mass is due to hypertrophy of granulosa and theca cells as well as some mitotic division of the latter. Also, there is a rapid mitotic division and growth of endothelial cells and fibroblasts. As measured by ⁸⁵ rypton clearance, the mature ovine corpus luteum has a high capillary blood flow, on the order of 1 ml · g¹ · min¹, in keeping with its high metabolic activity (177). In addition to connective tissue fibrocytes and endothelial cells lining capillaries, most species have two types of steroidogenic cells, both of which secrete progesterone. The smaller steroidogenic cells (<20 µm) are thought to be derived from the theca interna, and the large steroidogenic cells (20-30 µm) are thought to be derived from the granulosa cells lining the follicle wall (12). There is also evidence that at least some small steroidogenic cells may be transformed into large steroidogenic cells as the corpus luteum matures (12, 195). The small steroidogenic cells secrete low basal levels of progesterone but respond to LH with an increase in progesterone production, whereas large steroidogenic cells secrete high basal levels of progesterone but are unresponsive to LH stimulation (204, 382). Two types of steroidogenic luteal cells have been identified in the corpus luteum of sheep (204), cow (687), sow (413), rat (635), rabbit (324), rhesus monkey (307), and human (528). However, in the mare, in which ovulation occurs into an ovulation fossa and which develops secondary corpora lutea during pregnancy, it appears that only the granulosa lutein cells contribute to the formation of the mature corpus luteum (83). The large steroidogenic cells of ruminants possess secretory granules containing oxytocin that may contribute to the luteolytic process (see sects. III and IV). The cellular composition of the ovine corpus luteum is shown diagrammatically in Figure 3. It should be noted that, although the large steroidogenic cells constitute only 4% of the total number of cells, they constitute 25% of the cellular volume of the corpus luteum (195, 586).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Major cell types in ovine corpus luteum. Relative number of cells (%) and relative cell volume (%) of each cell type in corpus luteum are shown diagrammatically. Remaining number of cells (7%) are composed of plasma cells, lymphocytes, and granular leukocytes. Residual tissue volume (33%) is occupied by vascular lumina and intercellular spaces. Granulosa-derived large luteal cell constitutes 4% by number but 25% by volume of gland. [Based on data from Rodgers et al. (586) and Farin et al. (195).]

In contrast to nonprimate species, large and small steroidogenic cell types in the monkey are responsive to LH and CG stimulation in vitro, but as the luteal phase progresses, only the large cells are responsive (307). Human large and small steroidogenic cells also show different characteristics in that basal progesterone produced by large cells is only twice that of small cells (528), whereas in monkeys and sheep, the large cells produce more than 10 times the amount of progesterone as the small cells. Thus it is apparent that considerable differences exist among species as to the specific characteristics of large and small steroidogenic cells. More detailed information on the characteristics of large and small steroidogenic cell types in different species has been reviewed previously (227, 655).

	II.  LUTEOLYSIS

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A physiological explanation for the cyclical regression of the corpus luteum, which is observed most clearly in the group of mammals showing spontaneous ovulation and corpus luteum formation, proved to be elusive. Initially, because of the emphasis placed on the importance of pituitary support for the maintenance of the corpus luteum, it was considered that withdrawal of pituitary luteotrophins such as LH and/or prolactin might cause the cyclical regression of the corpus luteum. However, with the advent of more sophisticated methods for measuring circulating pituitary gonadotropins, it became apparent that, at the time of corpus luteum regression, measurable levels of these hormones were present in ruminants (455, 729) and primates (40, 329, 482). Thus it appeared that complete withdrawal of pituitary gonadotropins from the circulating blood was unlikely to be a general mechanism for the induction of corpus luteum regression. Moreover, in primates, the administration of GnRH pulses or LH itself does not prevent the timely regression of the corpus luteum (746). Thus not only is there no evidence for a complete withdrawal of gonadotropins, but even methods which sustain basal gonadotropin levels do not prevent luteolysis.

The importance of the uterus in the control of corpus luteum regression was first reported by Loeb (422, 423), who demonstrated that hysterectomy in the cyclic guinea pig abolished cycles and caused abnormal persistence of the corpora lutea. Similar effects were subsequently observed in the cyclic sheep, cow, pig, and mare and in the pseudopregnant hamster, rabbit, and rat [for a review on this topic, see Anderson et al. (21)]. In primates, however, hysterectomy has no effect on the length of the ovarian cycle or the life span of the corpus luteum either in women (52, 56, 166, 422, 480) or in monkeys (89, 103, 298, 516). The persistence of corpora lutea in nonprimate species after hysterectomy suggested that the uterus produced a substance that caused the cyclical regression of corpora lutea, although this view was not universal (508, 522).

Unlike primates, all species that show corpus luteum maintenance after hysterectomy possess a bicornuate uterus. In most of these species, a unilateral effect of hysterectomy was demonstrated, i.e., removal of one uterine horn causes maintenance of corpora lutea in the ovary on the side on which hemihysterectomy was performed (21, 245, 452, 455). That surgery per se caused the observed effects of hemihysterectomy was ruled out by observations made in sheep, cows, and guinea pigs with congenital absence of one uterine horn (21, 68, 457). These subjects showed a persistence of the corpora lutea in the ovary on the side where the uterine horn was absent, provided that vascular connections with the remaining uterine horn were also absent (531). In contrast, in women with congenital absence of the uterus, no marked alteration in ovarian cyclicity was observed (84, 127, 137, 224, 474), thus supporting the finding that hysterectomy does not influence luteal function in primates.

B.  Identification of PGF₂ as the Mammalian Luteolysin

1.  Sheep

The sheep has long been used as a model animal for the study of luteolysis, partly because the effects of hysterectomy on corpus luteum function are very pronounced and partly because the size of the sheep permits the collection of amounts of ovarian and uterine venous blood adequate for repetitive hormonal measurements (144, 158, 501, 623). An important advance in understanding the role of the uterus in the regulation of luteolysis came with the development of the ovarian autotransplant model at the Worcester Foundation in 1966 (252, 253). Such a development was a logical extension of the use of ex vivo organ perfusion systems (302, 588, 715) and tissue superfusion (667) for the elucidation of steroid biosynthetic pathways as pioneered at the Worcester Foundation. After the success of the adrenal autotransplant model at the Howard Florey Institute in Melbourne, Australia (475), Gregory Pincus had the foresight to establish the ovarian autotransplant model at the Worcester Foundation, thanks to the collaboration and surgical skills of Dr. James Goding from the Melbourne adrenal group. The ovarian autotransplantation technique involves the excision of the ovary with its vascular pedicle and transplanting it with vascular anastomoses of the ovarian artery and the utero-ovarian vein into a jugulocarotid skin loop (Fig. 4). The remaining ovary is excised and discarded with the uterus remaining in situ (252, 253, 455). Such a model permits long-term access to the arterial and venous supply of the ovary in the conscious, unstressed animal. Substances of interest can be infused directly into the arterial supply of the ovary, and frequent samples of ovarian venous blood can be obtained readily (455, 473). Moreover, ovarian blood flow can be determined by timing the collection of a given volume of ovarian venous blood (Fig. 4). After measuring the concentration of a specific hormone in ovarian venous plasma, the direct ovarian secretion rate of the hormone can then be calculated as mass per unit time (253, 473). However, animals with ovarian autotransplants showed a prolonged luteal phase (>100 days) apparently because the ovary in the jugulocarotid loop was separated from the uterus in the abdomen. Such an observation indicated that the putative control of luteal regression by the uterus in the sheep could not be mediated via the systemic circulation. Although the ovarian autotransplant model proved valuable for investigating the direct intraovarian effect of pituitary gonadotropins on ovarian steroid secretion in vivo (473), the lack of regular ovarian cyclicity was a disadvantage. Therefore, a method for autotransplanting the uterus and ovary together as one block of tissue to jugulocarotid loops was developed (455, 458, 465). This procedure was based on a method for transplanting the uterus and ovary subcutaneously with vascular anastomoses to the carotid artery and jugular vein (288). When one uterine horn and its adjacent ovary were transplanted with vascular anastomoses to a jugulocarotid loop, regression of the corpus luteum occurred at the expected time and steroid secretion, and LH levels were similar to the normal cycling intact sheep (455, 458, 465). These transplantation studies not only supported previous findings in utero-ovarian relationships (21), but proved unequivocally in the sheep that the uterus and ovary had to be contiguous for the occurrence of cyclic regression of the corpus luteum and for a normal cyclic pattern of hormone secretion. Cross-circulation experiments between ovarian and utero-ovarian transplants indicated that uterine venous blood possessed luteolytic activity at the time of corpus luteum regression (451, 452, 455, 456). This finding was supported by evidence that ovine uterine venous plasma, collected at the time of corpus luteum regression, had a progesterone-suppressing effect when infused into an ovarian artery (42, 96).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Diagram of technique for intra-arterial infusion of autotransplanted ovary in sheep and periodic collection of ovarian venous blood. With inflation of pneumatic cuff above carotid arterial pressure, carotid arterial blood containing an infusate supplies ovary. [From McCracken et al. (473), reproduced by permission of the Society for Endocrinology.]

During the course of these cross-circulation experiments, Pharriss and Wyngarden (552) proposed that PGF₂ might fulfill the role of a uterine luteolysin because it was relatively abundant in the uterus (174, 553) and was a potent venoconstrictor (167). Pharriss and Wyngarden (552) showed that large amounts of PGF₂ injected into rats caused a shortening of pseudopregnancy and a reduction of the progesterone content of corpora lutea. When PGF₂ was infused into the arterial supply of the transplanted ovary in the sheep (Fig. 5), the results mimicked the luteolytic factor in uterine vein blood in that corpus luteum regression occurred and a new ovarian cycle was initiated (451, 459, 466). In addition, when PGF₂ was infused systemically, no effect on corpus luteum function was observed in these animals (451). The negative results of systemic infusions of PGF₂ were explained partly by a dilution effect and partly by the rapid clearance of PG from the blood after one passage through the lungs in some species (199, 555). This latter observation strengthened the view that PGF₂ was the luteolytic hormone released periodically from the uterus and capable of acting locally on the adjacent ovary to cause corpus luteum regression. The manner in which PGF₂ from the uterus might reach the ovary without passing through the systemic circulation clearly had to be investigated.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Progesterone secretion rate and peripheral plasma levels of LH following intra-arterial infusion of PGF2 (2.5 µg · h1 · 6 h1) into autotransplanted ovary of sheep during luteal phase. This infusion rate was minimal effective dose required to induce luteolysis when given as a constant infusion.

Previous studies involving ligation of uterine and ovarian blood vessels had indicated the importance of a vascular pathway for the local effect of the uterus on ovarian function in several species (39, 245, 456). In sheep, the ovarian artery is closely attached to the surface of the utero-ovarian vein and transverses the vein in an extremely tortuous manner before entering the hilus of the ovary (Fig. 6). This curious vascular anatomy had been suspected previously of having functional importance at least in the cow (694). The possibility was considered that substances might diffuse from the utero-ovarian vein into the ovarian artery and reach the ovary directly without passing through the systemic circulation. Early evidence that direct local connections might exist between the ovary and the uterus was recorded in the giant fruit bat where unilateral hypertrophy of the uterine horn next to the ovary containing a newly formed corpus luteum was observed (445). In this case, it appeared that greater quantities of ovarian hormones reached the uterus from the ovary than would reach the uterus via the general circulation.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Anatomic relationship of ovarian artery and utero-ovarian vein in sheep and experimental design used to demonstrate countercurrent transfer mechanism. [From McCracken et al. (459). Reprinted with permission from Nature; Copyright 1972 Macmillan Magazines Limited.]

The initial demonstration of a countercurrent transfer mechanism in the ovarian vascular pedicle involved the transfer of [³H]PGF₂ from the uterine vein to the ovarian artery in the sheep (455). Later studies indicated that ~1% of [³H]PGF₂ infused into a uterine vein appeared in the adjacent ovarian artery after a lag of 20-30 min (see Fig. 7) (459). Thus the presence of a luteolytic factor in uterine vein blood at the time of luteolysis had been identified that was mimicked by infusions of PGF₂ into the arterial supply of the ovary. Moreover, a pathway had been found whereby a small portion of PGF₂ from the uterus (~1%) could bypass the systemic circulation and reach the ovary directly. It remained to be determined whether PGF₂ was secreted by the uterus into the uterine vein during the course of luteolysis in amounts large enough to have a luteolytic effect after local transfer to the ovary. This was achieved when PGF₂ was measured by gas chromatography/mass spectrometry (GC/MS) (267, 452, 455, 459) and by rat fundal strip bioassay (69) in samples of uterine vein plasma from a series of individual sheep around the time of corpus luteum regression. From these results, it was calculated that during luteolysis in the sheep, each uterine horn secreted 25 µg PGF₂/h into the uterine vein. When this quantity of PGF₂ was infused into a uterine vein in situ, premature corpus luteum regression was induced consistently in the adjacent ovary (459, 675), but no effect on the corpus luteum was observed when the same amount of PGF₂ was administered into the peripheral circulation (451). These infusion experiments not only confirmed the countercurrent transfer process in the utero-ovarian vascular pedicle, but also established the role of PGF₂ as a uterine luteolytic hormone in the sheep (459).

View larger version (37K): [in this window] [in a new window]   Fig. 7. Time course of countercurrent transfer of [3H]PGF2 from utero-ovarian vein to adjacent ovarian artery. [From McCracken et al. (459). Reprinted with permission from Nature; Copyright 1972 Macmillan Magazines Limited.]

As shown in Figure 8, GC/MS analysis of ovine uterine vein blood collected frequently over the time of luteolysis indicated that PGF₂ was secreted by the uterus as a series of pulses each lasting ~1 h and occurring at intervals of ~6-9 h (45, 456). Similar pulses of PGF₂ were also observed during luteolysis when measured by radioimmunoassay (RIA) (41, 673). Additional evidence for the pulsatile release of PGF₂ from the ovine uterus during luteolysis was obtained by the measurement of the primary metabolite of PGF₂, 15-keto-13,14-dihydro-PGF₂ (PGFM), in peripheral blood of sheep (373, 549). Pulses of PGFM occurred in the peripheral blood with a similar frequency to PGF₂ measured in uterine vein blood. The role of PGF₂ as a uterine luteolytic hormone was supported by the finding that systemic administration of indomethacin, an inhibitor of PG synthesis (527, 561), or the intrauterine administration of indomethacin (416) delayed or prevented luteal regression in several species. Also, immunization against PGF₂, either passively (193, 194) or actively (589, 600), delayed regression of the corpus luteum in the sheep.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Concentration of PGF2, progesterone, estradiol-17, and LH in utero-ovarian vein plasma sampled every 2 h during luteolysis in a sheep with a utero-ovarian autotransplant. Preovulatory rise of estradiol concentration is muted because of concomitant 5- to 10-fold increase in uterine blood flow. [From Barcikowski et al. (45); © The Endocrine Society.]

The proposed countercurrent mechanism for the transfer of PGF₂ in the utero-ovarian pedicle of the sheep initially met with some skepticism. One report by Coudert et al. (126) failed to confirm the transfer of [³H]PGF₂ in the uterovascular pedicle of the sheep, although in a companion paper, a transfer of xenon gas was demonstrated (125). However, at this time, it was also shown that unlabeled PGF₂ infused into the uterus of the cow caused a persistent elevation of PGF₂ in ovarian arterial plasma, but not in carotid arterial plasma (313), which supported the existence of a countercurrent mechanism. The countercurrent transfer of [³H]PGF₂ was confirmed subsequently by another group using the sheep (398). It was shown that the percentage of PGF₂ transferred from the uterine vein to the ovarian artery was mass dependent. This explained why Coudert et al. (125) failed to demonstrate a transfer of PGF₂, since the use of unlabeled PGF₂ as a carrier decreased the transfer of [³H]PGF₂ below the limit of detection of their method (0.7%). In sheep and cattle, experiments involving the anastomosis of the uterine vein or the ovarian artery in the hemihysterectomized animal have provided good support for the local transport of uterine luteolysin to the ovary (245). However, a large quantity of PGF₂ given by the systemic route causes luteolysis because enough PGF₂ escapes metabolism by the lungs to act on the ovary (456). Moreover, in many species including the sheep, cow, and pig, the corpus luteum is resistant to PGF₂ during the early part of the luteal phase (see below under different species).

An anatomic study of the utero-ovarian blood vessels and lymphatics in eight sheep demonstrated that, although no direct lymphatic connections existed between the uterus and the ovary, some uterine lymphatics were observed to lie adjacent to the ovarian artery in a fashion similar to the utero-ovarian vein. It was suggested that the uterine lymphatics might also serve as a possible route of transfer for uterine products to the ovary (113). Later studies in the sheep indicated that both the vena tubarius (15) and uterine lymph (2) contained a relatively high concentration of PGF₂, indicating that these two structures may also participate in the transfer process. Indeed, it was later shown that [³H]PGF₂, given as a single injection into the uterine lumen of sheep, reached a peak in a uterine lymphatic in 50 min while peak radioactivity in the adjacent uterine vein was achieved within a few minutes (295). Also, in this study, it was shown that the injection of [³H]PGF₂ into the uterine lumen resulted in a transfer of radioactivity to both the adjacent and, to a lesser extent, the opposite ovary (295). However, because [³H]PGF₂ was not measured in ovarian arterial plasma but rather in ovarian venous plasma after passing through the ovary, the dynamics of PGF₂ transfer into the ovarian artery were not established in this study. Receptors for PGF₂ are abundant in the ovary, and these may have bound a portion of the infused [³H]PGF₂, thus causing an underestimate of the percent of transfer of [³H]PGF₂ in this study (0.3%). Moreover, the lymphatics adjacent to the cannulated lymph vessel were ligated, which most likely concentrated the flow of lymph from the uterus into the utero-ovarian vascular pedicle (295). Nonetheless, the observed slower transport of PGF₂ from the uterine lumen into the uterine lymphatics compared with its rapid transfer into the uterine vein suggests that lymphatic transport may serve to extend the duration over which a pulse of PGF₂ secreted by the uterus acts at the ovarian level in this species.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Concentration of 15-keto-13,14-dihydroprostaglandin F2 metabolite (PGFM) in peripheral plasma of cyclic cow during luteolysis. Pulses of PGFM representing uterine secretion of PGF2 coincide with decline of progesterone during luteolysis. [From Kindahl et al. (370), Copyright 1984, with permission from Elsevier Science.]

Other studies in the sheep revealed that progesterone and several other ovarian steroids are transferred from the ovarian vein to the ovarian artery, indicating the presence of an ovarian-ovarian countercurrent system in this species (178, 462, 469). Thus it would appear that the sheep has two types of countercurrent systems in the vasculature of the reproductive system: 1) a utero-ovarian countercurrent system for the transfer of PGF₂ from the uterus to the ovary and 2) an ovarian-ovarian countercurrent system for the transfer of ovarian steroids back to the ovary and the Fallopian tube and probably also to the uterus. A countercurrent mechanism has also been identified in the human ovarian vascular pedicle (57, 58), although the evidence indicates an ovarian-ovarian countercurrent transfer with no evidence of a utero-ovarian transfer. Such an anatomic difference in the ovarian vascular pedicle in the human may explain, at least in part, why the effects of hysterectomy on corpus luteum function differ in the primate from those in nonprimate species.

Initially, a low-molecular-weight protein was proposed as the luteolytic substance in the cow (429), a view which was later modified to include arachidonic acid (618, 619, 285). However, other evidence indicated that, in the cow, PGF₂ is the luteolytic hormone that is released cyclically from the uterus at the time of corpus luteum regression. Prostaglandin F₂ is elevated as a series of pulses in uterine venous blood during luteolysis in this species (509). Also, it has been demonstrated that levels of PGFM in the peripheral blood of cows show pulsatile increases during luteolysis (371, 372, 374), thus supporting the role of PGF₂ as a luteolytic hormone (Fig. 9). Wild ruminants such as the reindeer also show a pulsatile release of PGF₂ during luteolysis (590) (see Fig. 10).

View larger version (21K): [in this window] [in a new window]   Fig. 10. Concentration of progesterone (dotted line) and PGFM (solid line) in peripheral plasma of reindeer during luteolysis. Horizontal axis shows time in hours. [From Ropstad et al. (590).]

A countercurrent mechanism for the transfer of PGF₂ appears to exist in the utero-ovarian pedicle of the cow (313). Prostaglandin F₂ injected into the uterus caused an elevation of PGF₂ in ovarian arterial plasma for several hours after PGF₂ levels had subsided in the jugular vein and carotid artery. However, a transfer of endogenous PGF₂ in the uterine horn to the ovarian artery in the cow following systemic challenges of oxytocin was not demonstrated (485). The authors point out that they took no control samples of peripheral arterial blood and in addition were unable to show a difference in concentration of PGF₂ between venous and arterial plasma, both of which gave readings of ~200 pg/ml. Further studies in the cow confirmed that a countercurrent transfer of PGF₂ occurs in this species (670). This was achieved by demonstrating a concentration gradient of PGF₂ between an ovarian artery and a peripheral artery. There is also evidence that PGF₂ may act, in part, through the systemic circulation in the cow, since normal cycles were observed in beef cattle after hemihysterectomy regardless of which ovary contained a corpus luteum (698). Moreover, when the ovary was transplanted with vascular anastomoses to the carotid artery and the jugular vein in a cow, and the other ovary was removed, estrous cycles were of normal duration (46). When one ovary was transplanted in the cow, the intact uterus with both uterine horns was left in situ in the abdomen and the other ovary was removed. Thus PGF₂ secreted by the whole intact uterus during luteolysis would produce a peripheral level of PGF₂ approximately twice that of the hemihysterectomized cow. The conclusion that PGF₂ may act partly via the systemic circulation in the cow is supported by the finding that only 65% of [³H]PGF₂ is metabolized in one passage through the lungs in the cow (138), whereas in the sheep, >99% is metabolized (139). Some of the apparent discrepancies concerning the local countercurrent transfer versus the systemic transport of PGF₂ in the cow may be related to the use of different breeds of cattle or to the amount of uterine tissue removed during hemihysterectomy. In the alpaca, a member of the camel family indigenous to South America, there is evidence that the luteolytic effect of the uterus is mediated locally in the right uterine horn but is mediated both systemically and locally in the left uterine horn (198).

Early work on the effect of hysterectomy on luteal function in the sow was carried out by du Mesnil du Buisson and Dauzier (169), who showed that bilateral regression of the corpora lutea in both ovaries of this polyovulatory species occurred after hemihysterectomy. However, if the amount of tissue in the remaining horn was reduced to ~25%, the corpora lutea in the opposite ovary no longer regressed (170). This indicated that there may be both a local and a systemic effect of the uterus on the corpora lutea in the sow. Indeed, in the reproductive tract of the sow, there is now anatomic evidence that a crossover of the venous drainage between both uterine horns may occur (148). Subsequently, PGF₂ was identified and measured by RIA in uterine vein blood of the sow at luteolysis as shown in Figure 11 (251). A local effect of PGF₂ in the luteolytic process in the sow was shown when luteolysis was induced by infusions of PGF₂ into an adjacent uterine vein (251), although there was some evidence that the opposite ovary was also affected. This may be caused by some of the infused PGF₂ reaching the opposite ovary either systemically (388) or by lymphatic connections with the opposite uterine horn (385). It is likely that PGF₂ can act in part via the systemic circulation, since ~40% of [³H]PGF₂ infused into the pulmonary artery traverses the lungs unchanged (139). This finding explains the evidence for both a local and systemic luteolytic action of PGF₂ in the sow. Bazer and colleagues (50, 494, 495) have presented additional evidence that PGF₂ is the luteolysin in the sow and have suggested also that the reduction in uterine PGF₂ secretion in early pregnancy is caused by a change in the uterus from an endocrine function to an exocrine function so that PGF₂ is secreted into the uterine lumen (50). It has been known for some time that exogenous estrogen, rather than shortening the cycle as in other species, appears to prolong the life span of the corpora lutea in the pig (235).

View larger version (29K): [in this window] [in a new window]   Fig. 11. Concentration of PGF2 and progesterone in utero-ovarian vein plasma and concentration of progesterone in peripheral plasma during luteolysis in sow. [From Gleeson et al. (251), Copyright 1974, with permission from Elsevier Science.]

Bazer and co-workers (219) have proposed that endogenous estrogen secreted during early pregnancy may be responsible for inhibition of luteolysis by switching the secretion of PGF₂ from the venous side of the uterine circulation to the uterine lumen (endocrine vs. exocrine). In the peripheral blood of sows, PGFM is elevated as a series of pulses at the time of luteolysis, a finding which serves to confirm the role of PGF₂ as a luteolytic hormone in this species (374). An unusual feature of luteolysis in the sow is that corpora lutea are refractory to exogenous PGF₂ for about the first 10-12 days of the 20- to 21-day cycle (156), although PGF₂ may be active earlier in the cycle if infused into the adjacent utero-ovarian vein (388), if a synthetic analog is used (274), or if repeated injections are given (187). The refractoriness to PGF₂ early in the cycle may be explained by the finding that the number of receptors for PGF₂ in the corpus luteum of the sow increases up to sevenfold as the luteal phase progresses (232). This increase did not occur in pregnant or estrogen-treated pigs (234). Moreover, these investigators suggested that uterine PGF₂ in the cyclic pig might cause the induction of its own receptor in the corpus luteum (187).

In the mare, there is evidence for a more dominant systemic effect of the uterus on the corpus luteum. Although total hysterectomy results in luteal maintenance (246, 649), hemihysterectomy failed to establish a unilateral relationship (246). Furthermore, in contrast to other species such as the sheep (161) and cow (333), where a much lower dose of PGF₂ is luteolytic when given by the intrauterine route versus the systemic circulation, both these routes of administration were equally effective in the mare (162). This is in keeping with the finding that the ovarian artery in the mare has very little direct contact with the uterine vein compared with the sheep and cow (244). Confirmation of the role of PGF₂ as a luteolytic hormone came from the measurement of PGF₂ in the uterine vein (163) and the elevation of PGFM in the peripheral blood (Fig. 12) at the time of luteolysis (514). The role of PGF₂ in the control of luteolysis in the mare has been reviewed previously (335, 613, 650, 745). In view of the lack of a local control of the ovary by the uterus in the mare, it is probable that, like the sow, a proportion of PGF₂ secreted by the uterus may escape metabolism by the lungs and thus may act systemically as a mediator of luteolysis.

View larger version (20K): [in this window] [in a new window]   Fig. 12. Concentration of PGFM and progesterone in peripheral plasma in mare during luteolysis. [From Stabenfeldt et al. (650).]

The reproductive pattern of the goat is rather similar to the sheep in many respects, but there are two important differences between these two species. First, the cycle length in the goat is longer at 19-21 days compared with 16-17 days in the sheep. Second, the goat relies exclusively on progesterone secreted by the corpus luteum for the maintenance of pregnancy, since the goat placenta, unlike the sheep, does not produce progesterone. As in the sheep, hysterectomy in the cyclic goat results in the maintenance of the corpus luteum (130). Prostaglandin F₂ is produced by the uterus in a pulsatile fashion and induces spontaneous luteolysis in this species (317, 370). Pulses of two metabolites of PGF₂ are observed on days 16-18 of the cycle, which coincides with the decline in peripheral plasma progesterone levels (Fig. 13). Parturition in the goat appears to involve a luteolytic mechanism, whereby PGF₂ secreted by the fetoplacental unit at term causes a decrease in progesterone production by the corpus luteum and, hence, contributes to the initiation of labor (131). Evidence for a role of PGF₂ in labor was demonstrated by the infusion of PGF₂ into a uterine vein adjacent to the corpus luteum of the pregnant goat, which caused regression of the corpus luteum and the induction of abortion or labor (129). Similarly, surgical removal of the corpus luteum before term in the goat results in abortion, an effect which could be prevented by the administration of progesterone (131). In a recent study, no increase in PGF₂ was detected before the preparturient decline in progesterone in goats (214). However, the authors point out that blood samples were collected only once daily before labor so that potential episodes of pulsatile PGF₂ secretion during preparturient luteolysis would not have been detected.

View larger version (34K): [in this window] [in a new window]   Fig. 13. Concentration of 2 metabolites of PGF2 and progesterone in peripheral plasma during estrous cycle and after mating in goat. Horizontal solid bar represents time of estrus, and 2 arrows indicate time of mating. [From Kindahl et al. (370).]

After the sheep, which, because of its size, offers substantial advantages as an experimental animal model for luteolysis, the strongest evidence for PGF₂ as a luteolytic hormone is found in the guinea pig (321, 561). The major pieces of evidence are 1) a luteolytic effect of administered PGF₂, 2) an elevation in PGF₂ levels in uterine venous blood at the time of spontaneous luteolysis, 3) prolonged cycles in animals treated with indomethacin or immunized against PGF₂, and 4) increased uterine PGF₂ secretion after treatment with exogenous estrogen. The correct vascular anatomy for a countercurrent system appears to be present in the guinea pig (147), which is in keeping with the known unilateral effect of the uterus on the life span of corpora lutea in this species. In addition, xenon-133 gas dissolved in saline is transferred locally from one uterine horn to the adjacent ovary in the guinea pig (175). Of several species examined, the clearance rate of labeled PGF₂ from the systemic circulation was most rapid in the guinea pig (262). Such a finding indicates that, as in the sheep, there is an absolute requirement for a local transfer of PGF₂ between the uterus and the ovary for luteolysis in the guinea pig. As in several other species, PGF₂ (measured as PGFM in peripheral blood) is secreted in a pulsatile fashion from the uterus of the guinea pig during luteolysis (Fig. 14) (183). Uterine PGF₂ production is suppressed during the establishment of pregnancy so that corpora lutea continue to secrete progesterone for the maintenance of pregnancy (562). The corpora lutea are necessary for the maintenance of pregnancy up until about day 28 when the placenta assumes this function (297).

View larger version (29K): [in this window] [in a new window]   Fig. 14. Concentration of PGFM and progesterone in peripheral plasma of guinea pig during luteolysis. Hourly blood samples were collected daily between hours of 9:00 A.M. and 4:00 P.M. [Adapted from Elger et al. (183).]

Hysterectomy is known to prolong the life span of the corpus luteum in the pseudopregnant rat (80), rabbit (25), and hamster (95), but not in the mouse (154). In addition, PGF₂ now has been shown to be luteolytic when administered in vivo to the pseudopregnant rat (552), rabbit (275), hamster (390), and Mongolian gerbil (160). Prostaglandin F₂ will also terminate pregnancy in the rat, rabbit (275), hamster (277, 391), and mouse (47). This last effect seems to be primarily a luteolytic one since exogenous progesterone prevents abortion in most of these species, although higher doses of PGF₂ were required to terminate pregnancy than to shorten pseudopregnancy in the mouse (47) and rat (111). The requirement for a luteolytic mechanism for the induction of labor in mice was demonstrated by the finding that mice rendered deficient in the gene for the PGF₂ receptor do not show the normal prepartum drop in progesterone and do not exhibit parturition [see Sugimoto et al. (660) and sect. VA]. Because of the small size of these experimental animals, there have been few direct measurements of levels of PGF₂ in blood. However, PGF₂ levels were found to be elevated in the peripheral blood of pseudopregnant hamsters at the time of luteolysis (610).

In the rabbit, in which luteal function is estrogen dependent, plasma progesterone levels begin to decline on day 14 of pseudopregnancy in both intact and hysterectomized animals, indicating that the initial decline of plasma progesterone does not depend on the uterus. On day 17, a marked drop in progesterone levels occurs only in intact pseudopregnant animals and is accompanied by an increase in uterine venous PGF₂ levels (432). In the rabbit, like the mare, there is evidence that the luteolytic effect of the uterus may be mediated systemically, since no unilateral effect of the uterus on the corpus luteum can be demonstrated (328). The importance of the systemic route is supported by the lack of an anatomic basis for a countercurrent transfer system between the uterus and the ovary (147) and by the absence of a local transfer of xenon-133 from the uterus to the ovary in the rabbit (175). The possibility that a metabolite of PGF₂ is the systemic component causing luteolysis is suggested by the finding that the metabolite 13,14-dihydro-PGF₂ was fourfold more luteolytic than PGF₂ when infused systemically in the pseudopregnant rabbit (354). Like the pig, there is evidence in the rabbit that estrogen, rather than hastening the onset of luteolysis, actually has a luteotropic action in the corpus luteum, possibly by protecting it against PGF₂ (276), or by changing the secretion of PGF₂ by the endometrium from an endocrine to an exocrine mode as proposed in the sow (494, 495).

As in most other species, basal levels of LH in the human appear to be essential to maintain the secretory function of the corpus luteum (690). In the rhesus monkey, bilateral lesions in the arcuate nucleus of the hypothalamus caused a cessation of ovarian ovulatory activity that could be restored by chronic circhoral infusions of GnRH (381). If plasma levels of LH were reduced to undetectable levels during the midluteal phase by halting GnRH infusions in these lesioned monkeys, plasma progesterone fell to undetectable levels. However, when LH levels were restored 3 days later by resuming circhoral GnRH infusions, the corpus luteum resumed a normal pattern of progesterone secretion but regressed at the expected time (330). These studies suggest that LH acts to promote progesterone synthesis by the corpus luteum but that other factors are responsible for the loss of function and structural integrity of the primate corpus luteum during luteolysis.

Because the primate corpus luteum undergoes luteolysis in the absence of the uterus, it has long been considered that luteolysis in primates might be an intraovarian event. Early studies suggested that estrogen produced by the primate corpus luteum mediated luteolysis (380). Subsequent work indicated that estrogen m