Abnormal Corpora Lutea Research Articles

Granulosa Cell-Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post-Ovulatory Follicle.

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post-ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC-layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post-ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)-cAMP-PKA-CREB signaling cascade, is necessary for mGC-layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC-layer stiffening", shedding light on the mechanism that prevents mGC escape from the post-ovulatory follicle.

Uterine Notch2 facilitates pregnancy recognition and corpus luteum maintenance via upregulating decidual Prl8a2.

The maternal recognition of pregnancy is a necessary prerequisite for gestation maintenance through prolonging the corpus luteum lifespan and ensuring progesterone production. In addition to pituitary prolactin and placental lactogens, decidual derived prolactin family members have been presumed to possess luteotropic effect. However, there was a lack of convincing evidence to support this hypothesis. Here, we unveiled an essential role of uterine Notch2 in pregnancy recognition and corpus luteum maintenance. Uterine-specific deletion of Notch2 did not affect female fertility. Nevertheless, the expression of decidual Prl8a2, a member of the prolactin family, was downregulated due to Notch2 ablation. Subsequently, we interrupted pituitary prolactin function to determine the luteotropic role of the decidua by employing the lipopolysaccharide-induced prolactin resistance model, or blocking the prolactin signaling by prolactin receptor-Fc fusion protein, or repressing pituitary prolactin release by dopamine receptor agonist bromocriptine, and found that Notch2-deficient females were more sensitive to these stresses and ended up in pregnancy loss resulting from abnormal corpus luteum function and insufficient serum progesterone level. Overexpression of Prl8a2 in Notch2 knockout mice rescued lipopolysaccharide-induced abortion, highlighting its luteotropic function. Further investigation adopting Rbpj knockout and DNMAML overexpression mouse models along with chromatin immunoprecipitation assay and luciferase analysis confirmed that Prl8a2 was regulated by the canonical Notch signaling. Collectively, our findings demonstrated that decidual prolactin members, under the control of uterine Notch signaling, assisted pituitary prolactin to sustain corpus luteum function and serum progesterone level during post-implantation phase, which was conducive to pregnancy recognition and maintenance.

LH Induces De Novo Cholesterol Biosynthesis via SREBP Activation in Granulosa Cells During Ovulation in Female Mice.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

In utero exposure to acetaminophen and ibuprofen leads to intergenerational accelerated reproductive aging in female mice

Nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesic drugs, such as acetaminophen (APAP), are frequently taken during pregnancy, even in combination. However, they can favour genital malformations in newborn boys and reproductive disorders in adults. Conversely, the consequences on postnatal ovarian development and female reproductive health after in utero exposure are unknown. Here, we found that in mice, in utero exposure to therapeutic doses of the APAP-ibuprofen combination during sex determination led to delayed meiosis entry and progression in female F1 embryonic germ cells. Consequently, follicular activation was reduced in postnatal ovaries through the AKT/FOXO3 pathway, leading in F2 animals to subfertility, accelerated ovarian aging with abnormal corpus luteum persistence, due to decreased apoptosis and increased AKT-mediated luteal cell survival. Our study suggests that administration of these drugs during the critical period of sex determination could lead in humans to adverse effects that might be passed to the offspring.

Serum biomarkers of tubal ectopic pregnancy: current candidates and future possibilities

Ectopic pregnancy remains a considerable cause of maternal morbidity and mortality worldwide. Currently, it is diagnosed using a combination of transvaginal ultrasound and serial serum beta-human chorionic gonadotrophin levels. Diagnosis is often delayed and these tests are time-consuming and costly, both psychologically to the patient and financially to health services. The development of a biomarker that can differentiate a tubal ectopic from an intrauterine implantation is therefore important. In the pre-genomic era, a one-by-one scientific approach has revealed over 20 candidate biomarkers that could be used as a test to diagnose ectopic pregnancy although at present their clinical utility is very limited. These biomarkers cluster into themes: markers of abnormal embryo/trophoblast growth, markers of abnormal corpus luteum function, markers of a growing pregnancy in the Fallopian tube, markers of inflammation and peritoneal irritation, and uterine markers of normal implantation. It is likely that this thematic approach will facilitate the identification of newer biomarkers using microarray technology and inform the development of investigative paradigms using multiple markers at the time of presentation.

The bovine dominant ovarian follicle

Central roles in reproductive biology (i.e., growth and development of the oocyte, steroidogenesis, and ovulation) are played by the ovarian dominant follicle (DF). The DF is different from other follicles because it can escape atresia (the fate of all other follicles), and if exposed to the LH surge, its cells will differentiate into the corpus luteum. The DF was originally studied by looking at the surface of ovary through a surgical approach. Current studies employ a less-invasive ultrasound technique to track the growth and development of the DF. Recruitment and selection, the processes that give rise to the DF, and dominance, the physiological state of the mature DF, are important areas of basic research. Results of these basic studies are easily translated into real-world problems in farm animal reproduction. Superovulation, for example, overrides the selection mechanism and increases the number of ovulations. Understanding the factors that affect the size of the recruited pool should increase success rates (i.e., number of collected embryos) for superovulation. In most animals, the DF is short-lived, existing for long enough to allow for the final maturation of the oocyte. Some DF become atretic because they mature during the luteal phase and are never exposed to the LH surge. For other DF, the LH surge redirects the DF toward its ultimate demise (i.e., luteinization, ovulation, and differentiation into the corpus luteum). The DF is managed pharmacologically within protocols for timed AI. When timed AI fails, there may be abnormal corpus luteum development and early embryonic loss; outcomes that are secondary to inadequate follicular cell maturation and incomplete oocyte capacitation in the DF. Future work on the DF will clarify its underlying biological functions so that a variety of needs in farm animal reproduction can be efficiently managed.

Incidence of Abnormal Corpus Luteum in Superovulated Ewes

In the present study, in order to investigate the incidence time of abnormal corpus luteum (CL) in superovulated ewes, CL formation was observed and plasma progesterone (P4) and estradiol-17β(E2) profiles were investigated during the non-breeding and breeding seasons in three breeds of ewes. The ewes were treated with a fluorogesterone acetate (FGA) sponge for 12 days, and a single injection of 20 mg pFSH on Day - 2 and 500 IU eCG on Day - 1 (FGA sponge removal: Day 0), for estrous synchronization and superovulation. All ewes were given an injection of 100 μg gonadotropin releasing hormone analogue (GnRH analogue) at estrous detection and inseminated with frozen-thawed semen by laparoscope on a fixed-time basis (32-36 h after FGA sponge removal). On Day 6.5, the ewes were laparotomized for ovarian response and embryo recovery. Blood was taken from all ewes on Days 0 to 6.5, and the plasma P4 and E2 concentrations were measured by emzyme immunoassay (EIA). According to the ovarian responses on Day 6.5, the ewes were divided into Group N (only normal CL; n=21), Group A (only abnormal CL; n=7) and Group M (both normal and abnormal CL; n=3). The mean P4 profile in Group N continued to rise during the period of blood sampling, and was significantly higher than in Group A which remained under 1 ng/ml and decreased after Days 4 to 4.5. The means E2 levels tended to be higher in Group A than in Group N. In the autumn study, the ewes with abnormal CL on Day 6.5 had already lacked in color on Day 4.5, and the number of large follicles was significantly lower in the ewes with abnormal CL than in those with only normal CL. These results indicate that the phenomenon of abnormal CL formation at 3-3.5 days after estrus is not due to premature luteal regression of functionally complete CL, but to luteal hypoplasia of incomplete CL with a short lifespan, but the cause of abnormal CL was not determined in the study.

Superovulation and embryo production in ewes using a commercial p-FSH

The effectiveness of a commercial porcine pituitary extract (p-FSH) with a 1:1 FSH/LH ratio was assessed on ovarian response and embryo yield in ewes. During the breeding season, 60 Altamurana ewes were synchronized with FGA-impregnated intravaginal sponges and subdivided into four treatment groups ( n = 15). Beginning 24 h before sponge removal, ewes were treated with 250 (Group 1), 500 (Group 2), 750 (Group 3) or 1000 IU p-FSH (Group 4) administered in a series of four decreasing doses over a period of 36 h. The ewes were monitored for estrus every 4 h, with ovarian response and embryos evaluated on the seventh day following sponge removal. No differences were found among the treatments in incidence of estrus and ovulation. Although treatment with 250 IU p-FSH produced the fewest ovulations, animals in this group yielded a higher ( P < 0.01) number of viable embryos, a lower ( P < 0.01) number of unfertilized ova, and a lower ( P < 0.05) incidence of unovulated follicles and abnormal corpora lutea when compared with animals treated with 750 or 1000 IU p-FSH, while treatment with 500 IU p-FSH yielded intermediate results. It is concluded that the commercial porcine pituitary extract (Pluset) at moderate doses (250 IU) is suitable for superovulation in ewes as it induces a high ovulatory response and transferable embryo yield.

Histological Characteristics of the Uterine Endometrium and Corpus Luteum during Early Embryogenesis and the Relationship to Embryonic Mortality in the Domestic Cat1

Pregnancy rates are low and litter sizes generally small when assisted reproduction techniques are used in gonadotropin-treated felid (cat) species. A prerequisite to determining whether or not abnormal morphological changes in the uterine lumen or corpus luteum (CL) are related to this reproductive failure is the documentation of normal histological kinetics during natural embryogenesis. This study characterized the histological changes of the endometrium and ovarian CL during the early stages of preimplantation embryonic development in the naturally estrous, mated queen. The purpose was to 1) develop a system for dating the cat endometrium and CL of early pregnancy; 2) document the frequency of abnormal uterine and CL history under natural mating conditions; and 3) compare histological traits of queens producing good- vs. poor-quality embryos. Naturally estrous, mated queens were ovariohysterectomized at 64 h (n = 8), 76 h (n = 11), 100 h (n = 8), 124 h (n = 7), 148 h (n = 6), or 480 h (n = 8) after first copulation. Embryos collected from oviductal and uterine flushings were graded for quality, and uteri and ovaries were fixed in formalin. Fixed tissue sections were stained and multiple histological traits described for each uterine (endometrial height, endometrial vacuolation, percentage of glandular cells with subnuclear vacuoles, number of mitoses, nuclear-to-cytoplasmic ratio) and ovarian (presence of tertiary follicles, vacuolation of luteal cell cytoplasm, luteal cell shape) sample. Modest histological changes were observed at each time point, and these were documented in detail. The most prominent modifications occurred at 124 h after first copulation and included thickening of the endometrium, straightening of the glands, increased cytoplasmic vacuolation, and increased epithelial height. Of 15 queens failing to produce good-quality embryos, only 4 expressed unusual histological characteristics; and 3 of 25 queens producing only high-quality embryos exhibited abnormal uterine or CL cellular integrity. Therefore, aberrant histological changes are not primarily responsible for failure of the naturally estrous, mated queen to produce good-quality embryos. Furthermore, a normative database now is available to date the endometrium and CL of early pregnancy and to examine the impact of exogenous gonadotropins and assisted techniques on uterine/CL structure and function.

Follicle stimulating hormone versus pregnant mare serum gonadotropin for superovulation of dairy goats

During the fall breeding season, 30 non-lactating mixed breed dairy goats were randomly allotted to one of three treatment groups. All does were implanted with 6 mg norgestomet for 11 d. Does in treatment A ( n = 10) received a single injection of 750 IU pregnant mare serum gonadotropin (PMSG) at the time of implant removal, while does in treatment B ( n = 10) were administered twice daily injections of 4, 3, 2 and 1 mg follicle stimulating hormone (FSH) given at 12 h intervals starting 48 h prior to implant removal. Control does ( n = 10) received only the norgestomet implant. Estrus was detected in 90% of the does in treatments A and B and in 70% of control does within 48 h of progestagen removal. Mean time to onset of estrus was similar for treatments A and B but was shorter ( P < 0.05) than time to onset for control does. Four does in treatment A returned to estrus within 7 d after the initial post-implant estrus. Both gonadotropin treatments produced more ( P < 0.01) corpora lutea (CL) than controls. The total number of CL was similar among treatments, but treatment A does had more ( P < 0.05) abnormal CL than does in treatment B. The number of ova recovered from does in treatments A and B was not different; however, the number of embryos recovered and the number of transferable quality embryos was less ( P < 0.01) for does in treatment A than in treatment B. These findings suggest that FSH is superior to PMSG for superovulation of non-lactating dairy goats during the breeding season.

Effects of progestagen treatment on concentrations of prostaglandins and oxytocin in plasma from the posterior vena cava of post-partum beef cows

The role of PGF-2 alpha in determining the lifespan of corpora lutea in the post-partum beef cow was investigated. In control cows (N = 5) induced to ovulate at Day 28 to 36 post partum by injection of 1000 i.u. hCG, corpora lutea had an average lifespan of only 8 days. In cows pretreated with 6 mg implants of a progestagen (norgestomet, N = 4) for 9 days, with implant removal 2 days before injection of hCG, luteal lifespan averaged 17.5 days. Concentrations of PGF-2 alpha in 9 hourly samples of plasma collected from the posterior vena cava via indwelling catheters were higher on Days 4 through 9 after injection of hCG (P less than 0.05) in the cows with short-lived corpora lutea. Greater release of PGF-2 alpha could therefore be a major factor in premature luteal regression. Concentrations of PGFM and oxytocin did not differ between cows with corpora lutea of short or normal lifespan. In a second experiment, concentrations of PGF-2 alpha in plasma from the posterior vena cava were examined during treatment with norgestomet (N = 8) or in contemporary controls (N = 7). In progestagen-treated cows, PGF-2 alpha was higher than in control cows (P less than 0.05), beginning on Day 3 of treatment and peaking on Day 5. It is concluded that the post-partum uterus increases secretion of PGF-2 alpha very early after first exposure to endogenous or exogenous progestagen.

Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe.

Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 micrograms) with (+P) and without (-P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the -P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes. All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and -P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the -P animals only exhibited a decline in progesterone production in vitro on Day 4 (P less than 0.01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the -P animals also decreased in weight (P less than 0.01) and progesterone content (P less than 0.001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only. These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.

The effect of restraint stress in early pregnancy in mice.

Mice were exposed to 5 h of restraint stress on Days 1-3, 4-6, or 1-6 of pregnancy in the morning (08:30-13:30 h, a.m.) or afternoon (13:30-18:30 h, p.m.). Stress reduced the pregnancy rate from 90 to 52% (P less than 0.005) and average litter size on Day 18 from 8.2 to 5.2 young (P less than 0.005). Stress for 6 days was more effective than for 3 days (P less than 0.005) and an a.m. stress was more effective than a p.m. stress (P less than 0.005) in reducing the average litter size. Animals examined on Day 7 after 6 days of a.m. stress had decreased numbers of normal corpora lutea (CL), increased numbers of abnormal CL, decreased serum progesterone concentrations and tended to have fewer implantation sites. Abnormalities of embryo transport and implantation were also present. Changes in CL morphology and embryo transport and development were evident on Day 4 after only 3 days of restraint stress. These results show that many reproductive events of early pregnancy can be disrupted by restraint stress.

Effects of progesterone (P 4) and estrogen (E 2) on fertilization failure and early embryonic mortality in superovulated cows

The effectiveness of a commercial porcine pituitary extract (p-FSH) with a 1:1 FSH/LH ratio was assessed on ovarian response and embryo yield in ewes. During the breeding season, 60 Altamurana ewes were synchronized with FGA-impregnated intravaginal sponges and subdivided into four treatment groups (n = 15). Beginning 24 h before sponge removal, ewes were treated with 250 (Group 1), 500 (Group 2), 750 (Group 3) or 1000 IU p-FSH (Group 4) administered in a series of four decreasing doses over a period of 36 h. The ewes were monitored for estrus every 4 h, with ovarian response and embryos evaluated on the seventh day following sponge removal. No differences were found among the treatments in incidence of estrus and ovulation. Although treatment with 250 IU p-FSH produced the fewest ovulations, animals in this group yielded a higher (P < 0.01) number of viable embryos, a lower (P < 0.01) number of unfertilized ova, and a lower (P < 0.05) incidence of unovulated follicles and abnormal corpora lutea when compared with animals treated with 750 or 1000 IU p-FSH, while treatment with 500 IU p-FSH yielded intermediate results. It is concluded that the commercial porcine pituitary extract (Pluset) at moderate doses (250 IU) is suitable for superovulation in ewes as it induces a high ovulatory response and transferable embryo yield.

Corpus luteum dysfunction and the epidemiology of breast cancer: a reconsideration.

Many of the epidemiologic risk factors for the development of breast cancer are related to major events in the female reproductive experience. Several years ago, in an attempt to synthesize these observations, we proposed a theory that focused on abnormal corpus luteum function as a major endocrine determinant of breast cancer risk. This review summarizes the work that has been done attempting to substantiate or refute that hypothesis and reconsiders it in the light of more recent information.

Anovulatory Menstrual Cycles in the Rhesus Monkey: The Significance of Serum Follicle-Stimulating Hormone/Luteinizing Hormone Ratios

Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol, and progesterone were determined for samples collected daily from seven mature rhesus monkeys during anovulatory menstrual cycles. Menstrual cycles in four monkeys were characterized by (1) low serum FSH concentrations, (2) a remarkably stable pattern of FSH secretion throughout the cycle, (3) low serum estradiol concentrations which varied little during the intermenstrual interval, and (4) low serum LH concentrations. Two monkeys had high serum FSH concentrations without the normal postmenstrual pattern of declining FSH concentrations to a preovulatory nadir. Estradiol concentrations increased progressively from 70 to 193 pg/ml over the first 18 days of the cycle in one monkey with high FSH levels and then sharply declined, suggesting follicular atresia. The seventh monkey had normal serum FSH levels, a normal pattern of FSH secretion, and normal FSH. LH ratios. The serum estradiol level in this animal increased from 62 to 327 pg/ml over the first 11 days of the menstrual cycle and then declined precipitously without a surge in gonadotropins, suggesting a functional deficiency at the hypothalamic or pituitary level. These data, taken in concert with our previous observations on abnormal corpus luteum function in the rhesus monkey, illustrate that the optimal growth, maturation, ovulation, and luteinization of the follicle are dependent upon (1) sufficient concentrations of both FSH and LH in the serum, (2) an appropriate pattern of FSH secretion, (3) an appropriate ratio of serum FSH:LH, and (4) an ovulatory surge of gonadotropins.

ADVANCEMENT OF OVULATION IN THE GUINEA-PIG WITH EXOGENOUS PROGESTERONE AND RELATED EFFECTS ON LENGTH OF THE OESTROUS CYCLE AND LIFE SPAN OF THE CORPUS LUTEUM

A single subcutaneous injection of progesterone (0-4 mg) was given to intact female guinea-pigs on either day 1, 7, 12, 13, 14, 15, or 16 of the behavioral oestrous cycle (day of oestrus is day 0). Injections given on either day 1 or day 7 had little effect, although there was a suggestion that injection on day 7 produced a lengthening of the cycle. Animals injected on either day 12 or day 13 underwent cycles of 16-9 and 18-1 days average length, respectively, which were significantly longer than the mean of 15-8 days for uninjected control females. Injection of progesterone on days 14, 15, or 16 was associated with one of three distinct sequelae: (1) simple prolongation of the cycle associated with a return to spontaneous oestrus 4--7 days later; (2) advancement of ovulation, formation of abnormal corpora lutea, and return to spontaneous oestrus 9--13 days later, and (3) return to spontaneous oestrus 14--16 days after the progesterone injection. These findings suggest that progesterone can cause the release of ovulatory amounts of gonadotropin following a period of endogenous oestrogen conditioning of the gonadotrophic system. If progesterone is administered before oestrogen conditioning is complete, then it inhibits or delays the conditioning process, and spontaneous oestrus and ovulation are postponed.

Fertility of Bovine Spermatozoa from Rete Testis, Cauda Epididymidis, and Ejaculated Semen

A total of 141 heifers and cows, predominantly Holsteins, was used to compare fertility of sperm entering and leaving the epididymis. Testicular and cauda epididymal sperm were obtained through indwelling cannulae while ejaculated sperm were collected before surgery on seven Holstein bulls. After deposition of 200×106 sperm in the uterine body, 106 females were killed on day 4 of a natural estrous cycle. Percentages of fertilized ova were 0% for testicular sperm, 84% for cauda epididymal sperm, and 94% for ejaculated sperm. Fertilizing capacity of cauda epididymal and ejaculated sperm was not significantly different. In contrast to observations for the other sperm types, testicular sperm were not detected on ova recovered on day 4. Testicular sperm also were not associated with five other ova, or in oviduct flushings, recovered 12 to 14h after uterine body inseminations. However, epididymal or ejaculated sperm were plentiful in ova and flushings from two cows killed 13 to 14h after insemination. The infertility of testicular sperm may result from intrinsic factors, a failure of sperm transport into the oviducts, or unusually rapid elimination of testicular sperm from the oviducts.Attempts to inseminate sperm surgically into the oviduct of 26 cows were unsatisfactory. Placement of sperm within the oviduct was uncertain with one technique while abnormal ovulation and/or corpus luteum formation, accompanied by a low incidence of ovum recovery, followed surgical inseminations by laparotomy.

Histology and Morphology of the Female Reproductive Tract of Ochotona princeps

Female pikas were collected from Mount Evans, Colorado. Weights of reproductive organs were recorded and the tissues were examined histologically and correlated with the specific phase of reproduction of the animal. From March through May, follicular growth, ovarian weight, and uterine weight indicated a probable estrogen response to increasing photoperiod, via the hypothalamicpituitary axis. The onset of behavioral estrus in May also correlated with probable estrogen increases. Formation of corpora lutea was identified in early June and was associated with increased vascularization of the uterus and the appearance of fetuses. Animals with resorbing fetuses possessed abnormal corpora lutea, suggesting a characteristic progesterone supported pregnancy. A striking difference between pikas and other lagomorphs, such as Oryctolagus cuniculus , was the functional corpora lutea during lactation. Histology of the oviduct and uterus were basically similar to that of Oryctolagus . Preimplantation fetal loss was low, indicating a high degree of reproductive efficiency.

A method for urinary pregnanediol determination using gas-liquid chromatography with electron capture

A gas-liquid Chromatographic method is described for urinary 5β-pregnane-3α, 20α-diol using 63Ni electron capture of the bis-heptafluorobutyrate as the end point This provided excellent sensitivity for menstrual cycle levels. Programmed automatic solid injection and automatic peak height integration improve productivity in pursuing the study of abnormal corpus luteum function.