Bovine Estrous Cycle Research Articles

Pituitary receptors for GnRH and estradiol, and pituitary content of gonadotropins in beef cows. I. Changes during the estrous cycle

To further characterize the endocrinological changes in the hypothalamo-hypophyseal axis thoughout the bovine estrous cycle, cycling beef heifers (n=24) were randomly assigned to six groups. These heifers were slaughtered 6, 12, 18, 19, 20 or 21 days following their previous estrus (day 0). Anterior pituitaries and hypothalami were collected. Hypothalami were divided into the preoptic area and medial basal hypothalamus, and content of gonadotropin-releasing hormone (GnRH) was quantified by radioimmunoassay. Contents of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the anterior pituitary gland were quantified by radioimmunoassay. Membrane receptors for GnRH were quantified by a standard curve technique and receptors for estradiol in anterior pituitary cytosol were quantified by saturation analysis. There was no significant change in content of GnRH in the hypothalamus or content of FSH in the anterior pituitary on any of the days examined; however, content of GnRH in the preoptic area was lower (P<.1) on day 19 postestrus. Cytosolic receptors for estradiol increased (P<.05) on day 18 post-estrus and returned to baseline by day 19. Content of LH and the number of receptors for GnRH in the anterior pituitary gland decreased (P<.01) on day 19 postestrus, and the number of receptors for GnRH remained low through day 21 postestrus. The reduction in anterior pituitary content of LH was transient indicating that synthesis of LH restores pituitary content to preovulatory levels before the number of receptors for GnRH returns to normal.

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle.

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle.

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.

Ovarian follicular growth, function and turnover in cattle: a review.

Studies in cattle assessing changes in number and size of antral follicles, concentrations of estradiol, androgens and progesterone in serum and follicular fluid, and numbers of gonadotropin receptors per follicle during repetitive estrous cycles and postpartum anestrus are reviewed. The rate of growth of small follicles (1 to 3 mm) into larger follicles increases as the estrous cycle progresses from d 1 to 18 (d 0 = estrus). Size of the largest antral follicle present on the ovary also increases with advancement of the estrous cycle. Most large follicles (greater than 10 mm) persist on the ovarian surface for 5 d or more between d 3 and 13 of the bovine estrous cycle. After d 13, most of these large follicles are replaced more frequently by new growing follicles (turnover) with an increased probability for recruitment of the ovulatory follicle after d 18. More research is needed to determine the time required for growth of bovine follicles from small to large antral size and evoke recruitment of the ovulatory follicle. Factors that regulate selection of the ovulatory follicle are unknown but may involve increased frequency of LH pulses in blood, altered blood flow and(or) changes in intrafollicular steroids and proteins. Quantitative evaluation of ovarian follicles indicated occurrence of consistent short-term changes in fluid estradiol and numbers of luteinizing hormone receptors in cells of large follicles only during the pre-ovulatory period. Presumably, low concentrations of follicular estradiol found during most of the estrous cycle are not due to a lack of aromatizable precursor or follicle-stimulating hormone receptors. Follicular fluid concentrations of progesterone increase only near the time of ovulation. Little is known about changes in follicular growth, turnover and function during postpartum anestrus in cattle. However, preliminary data suggest that the steroidogenic capacity of large follicles changes markedly during the postpartum period.

Differences in immunological and biological activity of serum luteinizing hormone during the bovine estrous cycle

Although immunological luteinizing hormone (LH) profiles have been extensively characterized in several species, an awareness has developed that these immunological LH measurements may not adequately describe the biological activity or stimulation of the target tissue. The objective of this study was to compare serum LH at different stages of the bovine estrous cycle as measured by both bioassay and radioimmunoassay. Blood samples were collected from 5 cows at 10 min intervals for 10 hr on days 3, 10 and 19 of the estrous cycle; for 24 hr at estrus on day 0 (natural preovulatory LH surge, NS); and for 8 hr following administration of luteinizing hormone-releasing hormone (induced LH surge) on day 10 of that cycle. Immunological activity of LH was determined by a previously validated radioimmunoassay. A sensitive and specific in vitro mouse Leydig cell bioassay in which testosterone production was an index of added LH was used to assess biological activity of LH. The same preparation (NIAMDD-bLH4) served as the standard for each assay system. Patterns of biological active LH closely followed those of immunological LH, although biological LH values tended to be lower. Although the pulse frequency (pulses/hr) of immunological LH was lower (P<.05) on day 10 ( .48 ± .07; x ± SE ) than day 3 or 19 (.88 ± .09 and .88 ± .15, respectively), no changes in biological LH pulse frequency were noted (P>.05). The mean biological to immunological (B:I) ratios were greater (P<.05) on days 3 and 10 (0.91 ± .16 and 0.93 ± .12, respectively) than day 19 (0.64 ± .05) and day 0 (0.43 ± .07). The B:I ratios of the natural LH surge and induced LH surge were not different from each other (P>.05). These observations demonstrate that biological LH exhibits a pattern similar to, but not identical to, immunological LH. Furthermore, as evidenced by changing B:I ratios, the pituitary releases LH which is not of the same biological activity at all stages of the estrous cycle. These data suggest that both quality and quantity of LH released may be important in regulating LH action at the target tissue.

Changes and interrelationships among luteal LH receptors, adenylate cyclase activity and phosphodiesterase activity during the bovine estrous cycle.

Changes and interrelationships among the cellular mechanisms that may regulate luteal progesterone synthesis during the bovine estrous cycle were studied. Corpora lutea (CL) were enucleated from 30 cows via a transvaginal incision on d 4, 7, 10, 13, 16 and 19 following estrus (estrus = d 0). Mean corpus luteum weight, and luteal progesterone and plasma progesterone concentrations increased (P less than .05) from d 4 to 7 or 10, and declined following luteal regression (d 19). Unoccupied LH receptor (UOR) concentrations increased (P less than .05) more than fourfold from d 4 (38 fmol/mg protein) to d 16 (173 fmol/mg protein) before declining (P less than .05) by d 19 (30 fmol/mg protein). The dissociation constant for UOR concentrations increased (P less than .05) during the estrous cycle. Concentrations of occupied LH receptors (OR) were less (P less than .05) on d 7 than other days; however, the total number of OR/CL increased fourfold from d 4 (47 fmol/CL) to d 10 (221 fmol/CL) and remained similar thereafter. Basal adenylate cyclase, LH-activated adenylate cyclase, and Gpp(NH)p-activated adenylate cyclase activities were greatest (P less than .05) on d 7 to 16 as compared with d 4 and 19. Luteinizing hormone stimulated (P less than .05) adenylate cyclase activity relative to basal activity on d 7, 10, 13, and 16; whereas, Gpp(NH)p stimulated (P less than .05) adenylate cyclase activity relative to basal activity at each period. Phosphodiesterase was 46% greater on d 19 as compared with d 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Column chromatography and electrophoresis of bovine uterine lumenal proteins collected during the estrous cycle

Uteri of 29 normally cycling Holstein and Jersey cows were non-surgically flushed with 50 ml sterile 1.5% saline, and fluids were recovered to evaluate biochemical methods for determination of qualitative changes in uterine lumenal protein at known stages of the estrous cycle. Total protein (mg) and number of red blood cells (million/ml) were 17.7 and 9.8; 7.6 and 7.1; 9.1 and 6.0; and 26.2 and 2.8 at day 0, 5, 10 and 15 of the bovine estrous cycle. Column chromatography (Sephacryl S-200) of uterine secretions revealed seven uterine specific peaks at ambient temperatures. One peak may be a hemoglobin contaminant. Five uterine specific protein peaks representing proteins greater than 160 000, ∼ 25 000 and less than 13 700 mol. wt. (3) were eluted with high performance liquid chromatography. Native polyacrylamide disc gel electrophoresis fractionated uterine fluid into as many as 13 bands. There were differences in six protein bands between uterine fluid and plasma. The consistency between Sephacryl S-200 and high performance liquid chromatography is the presence of three to four low molecular weight (< 13 700) uterine specific proteins. Sephacryl S-200 chromatography resulted in elucidation of a uterine specific protein approximately 60 000 mol. wt. not found with high performance liquid chromatography. However, proteins with mol. wt. > 160 000 and approximately 25 000 were found with high performance liquid chromatography. Results indicate no differences in protein class during the estrous cycle and that red blood cell contamination must be monitored during qualitative evaluation of uterine proteins.

Effect of charcoal-treated bovine follicular fluid on secretion of LH and FSH in ovariectomized heifers.

The objective of the experiment was to determine whether pools of charcoal-treated bovine follicular fluid (bFF) removed from large (greater than 10 mm) follicles during the preovulatory (d 18 to 1), postovulatory (d 2 to 6) or mid-cycle (d 7 to 17) stages of the bovine estrous cycle altered secretion of luteinizing hormone (LH) or follicle-stimulating hormone (FSH) in ovariectomized heifers. After a 20-ml injection of bFF given at three consecutive 6-h intervals, secretion of FSH was depressed from approximately 116 to 53 ng/ml by each pool of bFF. In contrast, bFF did not affect secretion of LH. Serum from an ovariectomized cow (control) had no effect on secretion of LH or FSH. We concluded that bFF contained a nonsteroidal component (perhaps inhibin) that suppressed FSH secretion in heifers.

Distribution of flehmen reactions of the bull throughout the bovine estrous cycle

A bull's flehmen reactions were recorded daily during 20-minute contact with 3 cows for nine months. During estrous periods (−3 to +1 days) there were 174 observations: 28.2% without flehmen, 21.2% with a single reaction, and 50.6% with repeated reactions. During nonestrous periods there were 624 observations: 49.0% without flehmen, 40.4% with a single reaction, and 10.6% with repeated reactions. Average numbers of all and repeated reactions during estrous periods were 2.11±2.30 and 1.90±2.43 respectively. Corresponding values for nonestrous periods, 0.70±0.93 and 0.29±0.93, were significantly lower (P<0.01). The results suggest that either the estrous pheromone(s) also occur(s) for a limited time or in limited amounts during nonestrus or that flehmen is not a very specific response to the estrus-related odors. In either case, the study of sexual pheromones should encompass other methods as well as flehmen observation.

Countercurrent transfer of testosterone by the ovarian vascular pedicle of the cow: evidence for a relationship to follicular steroidogenesis.

Utilizing an in vitro perfusion procedure, the current studies were performed to determine whether [3H]T testosterone ([3H]T) is transferred from ovarian vein to artery by countercurrent penetration during Days 18–19 of the bovine estrous cycle. The possible relationship of such a transfer to follicular steroidogenesis was also examined. Infusion of [3H]T, but not 51Cr-red blood cells, into the ovarian vein 1–2 cm below the ovarian hilus resulted in the recovery of radiolabel (ⵐ4.7%:range=2–1 3%) in ovarian arterial blood of all preparations tested. We also infused [3H]T directly into the arterial circulation. During constant-rate infusion, approximately 43% of the [3H]T infused into the vein and 37% of that infused into the ovarian artery remained sequestered within the periovarian tissue-fluid complex. At the end of 30-mm experiments, a markedly higher (P<0.001; P<0.05) amount of radioactivity per ml follicular fluid (FF) was observed in the largest follicle compared to that observed in small-medium follicles. Radioactivity remaining in follicles as testosterone (T) was markedly lower (P<0.0O1) and concentrations of estriol, dihydrotestosterone! androsterone, androstanedione, estradiol-17β, androstenedione, androstanediol, and estrone were consistently higher (P<0.O01) in follicular fluid (FF), granulosa (Gr) and theca (Th) relative to concentrations in the highly purified infusate (>99.5% T). In 60-mm experiments, in which perfusion continued for 30 mm after termination of [3H]T infusion, sequestered radiolabel as T declined (P<0.001) in both follicles (17–20%) andovarian venous effluent (2–5%) relative to that present in the infusate. Coincident with this decline in ([3H]T was a marked (P<O.001) increase of all seven metabolites studied in FF, Gr and Th. All of these steroids, except androstanediol and estradiol-17β, increased (P<0.05) in ovarian venous effluent of ovaries bearing the largest follicle or small-medium follicles. Results suggest an important countercurrent concentrating mechanism for T in the ovarian vascular pedicle which provides additional substrate for follicular metabolism/aromatization.

Effect of repeated laparoscopic surgery on the bovine estrous cycle

The effects of repeated laparoscopic surgery on the length of the bovine estrous cycle, estrus, ovulation and corpus luteum function were determined after one estrous cycle of normal duration (18 to 24 days). Five, Angus x Hereford cows were subjected to laparoscopy on days 5, 13, 18 and 20 (estrus = day 0) of the subsequent cycle. Blood was collected daily during the cycle in which laparoscopy was performed (surgical cycle) and during the next cycle (postsurgical cycle). Lengths of the surgical and postsurgical cycles (22.3 ± .5 days and 21.5 ± .6 days, respectively) did not differ (P>.05) from that of the presurgical cycle (21.8 ± .2 days). Average concentrations (ng/ml) of LH and progesterone in serum were similar during the surgical and postsurgical cycles (1.2 ± .1, 2.2 ± .2 vs 1.3 ± .2 and 2.3 ± .1). Progesterone concentrations remained above 1 ng/ml for 17 and 16 days during the surgical and postsurgical cycles, respectively. A pre-ovulatory rise in LH, along with estrus and ovulation was confirmed in all animals. Follicular development, characterized by follicular volume, increased progressively from days 5 to 20, with the largest increase occurring between days 13 and 18. These results indicate that laparoscopy, used at the times and frequency specified, does not alter reproductive function of cyclic cows and can provide information on ovarian activity.

Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle

In cows, electrocauterization of large follicles at the end of the luteal phase, lengthened the life span of corpora lutea. Injection of 5 mg of estradiol valerate or of 1 mg of an analogue of prostaglandin F 2α induced luteolysis; however, the injection of estrogen was associated with precocious estrus without either ovulation or corpus luteum growth. Injection of both estradiol valerate and prostaglandin analogue gave the same results as estradiol valerate alone. Deferred luteolysis, observed after electrocauterization of large follicles, seemed to be due to the withdrawal of estrogens and the consequent lack of prostaglandin F 2α production.

Accuracy of Predicting Stages of Bovine Estrous Cycle by Gross Appearance of the Corpus Luteum

Four readily identifiable changes in appearance of corpora lutea (Stages I to IV) occur during a bovine estrous cycle. Accuracy of estimating the stage of an estrous cycle by appearance of corpora lutea was determined in a double-blind study. One investigator observed estrus in a group of heifers while another with no prior knowledge of reproductive histories of the heifers estimated stage of the estrous cycle by visual inspection of their corpora lutea. Stage of the estrous cycle (Stage I, days 1 to 4; Stage II, days 5 to 10; Stage III, days 11 to 17; Stage IV, days 18 to 20) was estimated correctly in 41 of 48 heifers. The correlation between estimated and actual days of the estrous cycle was .81. In addition, concentrations of progestins and weights of corpora lutea during estimated stages of the estrous cycle were similar to many other investigations. Stages of the estrous cycle in heifers can be estimated from appearance of corpora lutea.

On the control of prostaglandin release during the bovine estrous cycle. Effects of progesterone implants

Progesterone implants were inserted subcutaneously on day 12 of the estrous cycle in three heifers. The implants were known to release an amount of progesterone sufficient to maintain the peripheral blood plasma at a level of about 1 ng/ml. All three heifers showed luteolysis in a similar way with peaks of 15-keto-13,14-dihydro-PGF 2α of normal magnitude and duration. Concomitantly, progesterone levels declined, but due to the presence of the implants, progesterone was maintained at a level of about 1 ng/ml. The release of prostaglandins, however, continued throughout the experimental period and did not cease until the implants were removed. It is concluded that prostaglandin release continues for as long as the progesterone levels are elevated, above a low and critical level.

Influence of Environmental Heat on Peripheral Plasma Progesterone and Cortisol During the Bovine Estrous Cycle

Plasma progesterone and Cortisol were measured in jugular blood by competitive protein-binding assay. Six cycling, estrus-synchronized Guernsey heifers were followed for four consecutive estrous cycles under two controlled temperature (18.2C, 55% relative humidity; and 33.5C, 55% relative humidity) conditions. Prolonged exposure to heat of Guernsey heifers increased plasma progesterone on days 2 to 19 of the first cycle and only on days 2 to 8 of the second cycle. However, heat stress depressed plasma Cortisol in both cycles and reduced the correlation coefficient between these steroids relative to specific stages of the estrous cycle.

Serum levels of follicle-stimulating hormone during the bovine estrous cycle.

Summary A specific radioimmunoassay for bovine follicle-stimulating hormone (bFSH) has been developed using rabbit antiserum to ovine FSH and radioiodinated human FSH. Employing this radioimmunoassay, as little as 3 ng of FSH can be quantified. Inhibition curves obtained with bovine serum, crude pituitary extract and other partially purified bovine pituitary preparations containing FSH were parallel to those obtained with the bFSH standard preparation. FSH estimates obtained by radioimmunoassay in preparations containing varying amounts of FSH, LH, TSH, GH and prolactin were in good agreement with the values obtained by bioassay. Exogenous bFSH could be quantitatively recovered from 200 ul of serum suggesting that serum components per se did not interfere with the radioimmunoassay. serum concentrations of FSH (in equivalents of NIH-FSH-S1) were: normal males, 76+ 5 ng/ml; castrated males 102 -+ 2 ng/ml; females in follicular or luteal phase 66 + 8 ng/ml and estrus females 78 +- 8 ng/ml. When serum samples were collected at daily intervals, there was a trend for FSH levels to be elevated on the day of estrus, however, this elevation was not significant. However, a significant increase in FSH concentrations was noted coincident to the LH peak when samples were collected at 2-hr. intervals around estrus. Following the injection of 250 ~ug of gonadotropin releasing

Jugular Plasma Concentrations of Alpha Amino Nitrogen, Amino Acids, Urea Nitrogen, and Glucose During the Bovine Estrous Cycle

Jugular plasma concentrations of alpha amino nitrogen, acidic and neutral amino acids, urea nitrogen, and glucose were measured during estrous cycles of 33 lactating cows. The estrous cycle was divided into four periods: proestrus (1 to 5 days before estrus), estrus, metestrus (1 to 3 days postestrus), and diestrus. Mean alpha amino nitrogen concentrations for the four periods were 4.85, 3.86, 4.06, and 4.53 mg/100ml. Acidic and neutral amino acids were assayed in samples drawn before and after the proestrus estrogen peak. Threonine, valine, methionine, isoleucine, and proline were all higher before than after the estrogen peak. Neither plasma urea nitrogen nor glucose concentrations varied significantly with different phases of the estrous cycle. However, the mean plasma glucose concentration during metestrus was 29.2% lower than during estrus. The hypoaminoacidemia at estrus may decrease availability of precursor for hepatic gluconeogenesis.

Serum progesterone and luteinizing hormone levels during the bovine estrous cycle.

Serum progesterone and LH levels were measured in the same serum samples at various stages of the estrous cycle in 7 cyclic heifers. Progesterone values were determined by gas liquid chromatography, and LH levels were measured by a solid phase radioimmunoassay, using an antiserum to bovine LH. The radioimmunoassay was specific for LH as judged by several criteria and serial dilutions of standards and unknowns gave parallel curves. Various tests indicated that immunoreactive LH as measured by the radioimmunoassay was related to biological activity. Most animals had basal levels of serum LH of 2–4 ng/ml. A preovulatory LH peak (7–50 ng/ml) was observed in every heifer and LH levels declined rapidly following this peak. Every heifer showed a small luteal phase LH peak and 5 of the 7 heifers showed another LH peak 4–7 days before ovulation. These fluctuations in basal LH levels may be correlated with growth and atresia of anovulatory follicles. Progesterone levels were very low following estrus (>100 ng/100 m...

Pituitary and hypothalamic endocrine changes during the bovine estrous cycle.

Pituitary and hypothalamic endocrine changes during the bovine estrous cycle.

Corpus luteum growth and progesterone synthesis during the bovine estrous cycle.

Luteal growth and steroidogenesis were studied in corpora lutea from 33 heifers, five each at 2, 4, 7, 11, 18 and 20 days after estrus, and three on the day of the next anticipated ovulation (day 0). Luteal weight and total DNA growth curves leveled at day 11. Ratios of RNA/DNA was greatest during most rapid luteal growth at day 4. Free sterol concentration in fresh corpora lutea increased linearly from 1,345 µg./gm. at day 2 to 2,612 µg./gm. at day 18, but esterified sterol decreased from 618 µg./gm. at day 2 to 344 µg./gm. at day 11, and then increased to 1,081 µg./gm. on the day of the next ovulation. Corpus luteum progesterone concentration was greatest at mid-cycle (46 µg./gm.). Incubation of homogenates of corpora lutea for 1 hr. in Krebs-Ringer bicarbonate buffer resulted in synthesis of progesterone which increased from 17 µg./gm. on day 2 to 136 µg./gm. on day 11 and 150 µg./gm. on day 20, but decreased to 46 µg./gm. on the day of the next ovulation. Total progestogen synthesis at any stage of the cvcle was no greater in the presence of NADPH than in the presence of NADP+glucose-6-phosphate. LH failed to stimulate synthesis of progesterone in these luteal homogenates. Significant quantities of esterified sterol accumulated during incubation of corpora lutea from day 18, 20 and 0, but not during incubation of younger tissues and rates of esterification of cholesterol-7-3H in the incubation medium were greater in the older than in the younger corpora lutea.