Characterization and distribution of protein kinase C in ovarian tissue

Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.     

Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development in the rat. II. Evaluation of the catalytic subunit inhibitor activity

In a previous report on the ontogeny of the ovarian adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity during prepubertal development of the rat, we concluded that the 4-fold decline in cAMP-dependent protein kinase activity observed in ovaries of 21- to 23-day-old rats was due to the presence of a heat-labile inhibitor in the ovarian extracts (Hunzicker-Dunn et al., 1984). We developed an assay for this ovarian kinase inhibitor activity that was based on the observation that ovarian cytosol added to an exogenous catalytic subunit of cAMP-dependent protein kinase caused a time-dependent and ovarian cytosol protein concentration-dependent inhibition of exogenous catalytic subunit phosphotransferase activity. The present studies were conducted to evaluate the basis for this catalytic subunit inhibitor present in soluble rat ovarian extracts of prepubertal-aged rats. This inhibitor activity was absent from cytosol extracts of rat corpora lutea, rat liver, rabbit follicles, and rabbit corpora lutea. Inhibitor activity present in rat ovarian cytosol was not attributable to insufficient levels of the phosphorylation substrate Kemptide. Inhibitor activity was also not related to the presence of the large amount of catalytic subunit-free regulatory subunit of the cAMP-dependent protein kinase present in ovarian extracts of late juvenile-aged rats. Inhibitor activity, however, did correlate with an endogenous adenosine triphosphatase (ATPase) activity that reduced assay ATP concentrations below levels needed to accurately measure phosphotransferase activity, despite the presence of sodium fluoride (an ATPase inhibitor) and ATP concentrations 5- to 15-fold greater than the Km of the kinase for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of pregnenolone synthesis by cytochrome P-450scc in mitochondria from porcine corpora lutea and granulosa cells of follicles

Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Biochemical changes in lipids during follicular growth and corpora lutea formation and regression in rat ovary

A biochemical study has been made on quantitative and qualitative changes in lipids of small (less than 300 microns), medium (300 to 550 microns) and large (less than 550 microns) follicles and in the fully developed and regressing corpora lutea. The total lipid content increased in the growing follicles and corpora lutea. Phospholipids formed the major component of total lipids in small sized follicles and developed corpus luteum. The cholesterol amount increased with the growth of follicles but decreased in the developed and regressing corpora lutea. Glycerides were the main fraction of total lipids in regressing corpora lutea. Free fatty acids were present in minor quantities in the growing follicles and corpora lutea. The physiological significance of these lipid changes is discussed.     

Regulation of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the bovine ovary in vivo and in vitro

In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. 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 were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen with no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective activation of rabbit ovarian protein kinase isozymes in rabbit ovarian follicles and corpora lutea

The magnitude of activation of the type I and type II forms of cAMP-dependent protein kinase was investigated in estrous follicles and corpora lutea (CL) obtained from ovaries of control rabbits and rabbits injected acutely with human chorionic gonadotropin (hCG). To this end, a chromatographic technique which permitted quantitative evaluation of the in vivo activational state of the two forms of cAmP-dependent protein kinase was developed and verified. Results revealed that in follicles obtained from ovaries of untreated estrous rabbits, 15% of the soluble cAMP-dependent protein kinase, all of which exists as the type II isozyme, is activated. Intravenous administration of a single bolus of hCG promoted a concentration-dependent activation (in 10 min) of this protein kinase isozyme. In CL obtained from ovaries of control, 4-day pseudopregnant rabbits, 32% of the total soluble cAMP-dependent protein kinase exists as the type I form and 68% exists as the type II form. Both types of protein kinase are approximately 10% dissociated in CL from ovaries of untreated rabbits. Upon intravenous administration of hCG, only the type I form of cAMP-dependent protein kinase is further activated (in 10 min). Dissociation of this protein kinase is dependent upon the time and concentration of hCG. Preferential activation of the type I form of cAMP-dependent protein kinase in CL is also demonstrable in in vitro studies using exogenous cAMP. These data suggest that the physiological intracellular mediator of acute cAMP-regulated, hCG-triggered functions in rabbit ovarian follicles is the type II isozyme of cAMP-dependent protein kinase while in CL of 4-day pseudopregnant rabbits, it is the type I enzyme form.     

Expression and redistribution of cellular Bad, Bax, and Bcl-X(L) protein is associated with VCD-induced ovotoxicity in rats.

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 +/- 0.16; mitochondria, 2.20 +/- 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-x(L) protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 +/- 0.08; cytosol, 1.39 +/- 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-x(L) in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 +/- 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-x(L) protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-x(L) ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.     

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.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

Uterine involution and ovarian changes during early post partum in autumn-lambing Corriedale ewes

The time of uterine involution and the changes in ovarian follicle populations were studied during early postpartum in multiparous, suckling Corriedale ewes lambing in the autumn. On Day 1 (n=5), Day 5 (n=4), Day 17 (n=4) and Day 30 (n=3) postpartum ewes underwent surgery to obtain ovaries and uteri. The weights of uteri and the lengths of the previous pregnant and nonpregnant horns were recorded as well as the presence of ovarian follicles larger than 1 mm in diameter. Uterine weight and length of uterine horns decreased (P2 to < 4 mm) follicles were also present. At days 17 and 30, aside from the small and medium follicles, all the ewes also had large (>/= 4 mm) follicles, and, at Day 30 2 ewes had large corpora lutea. We conclude that in autumn-lambing Corriedale ewes macroscopic uterine involution was complete around Day 17 post partum and that follicle development begins immediately after parturition, reaching preovulatory size before Day 17. In 2 of the 3 ewes studied until Day 30, ovulation (first progesterone increase) occurred after Day 17 (Days 18 and 25).     

Changes in follicular populations following treatment of buffaloes with PMSG (eCG) and Neutra-PMSG for superovulation.

Some 19 buffaloes were synchronized by administration of a prostaglandin (PG) salt Lutalyse, with a single intramuscular (i.m.) injection of 25 mg at day -13. Luteolysis was induced by administration of 50 mg PG, in divided doses of 30 and 20 mg i.m. 12 h apart on day 0 of experiment. The 30 mg PG injection was designated as 0 h of experiment. Group I animals (n = 6) received saline and served as controls while animals in Groups II (n = 7) and III (n = 6) received 2500 I.U. PMSG (eCG) i.m. at day -2. Group III animals were administered 5 ml Neutra-eCG intravenously at 60 h. The number of follicles, classified on the basis of diameter as small (2-5 mm), medium (6-9 mm) and large (> or = 10 mm) was assessed by ultrasonography on days -2, -1, 0, 1, 2, 5 and 7 of experiment. The number of corpora lutea (CL) was recorded by palpation per rectum on day 8. The number of small follicles which did not differ among the three groups on days 0, 1 and 2 was significantly lower (P < 0.05) in Group II animals compared to those in Groups I and III on days 5 and 7. The number of medium follicles increased after eCG treatment and was significantly higher (P < 0.05) in animals of Groups II and III on days 0 and 1, compared to control animals of Group I. It was, however, not different among the three groups on subsequent days of experiment. The number of large follicles which did not differ among the three groups on days -2, 0, 1 and 2 was significantly higher in Groups II (P < 0.01) and III (P < 0.05) animals compared to those of Group I on day 5. On day 7, the number of large follicles was in the order (P < 0.05) Group II > Group III > Group I. The number of CL in Group II animals was significantly higher (P < 0.05) than that in Group I animals but was not different from that of Group III animals. These results suggest that treatment of buffaloes with eCG for superovulation reduces the number of small follicles and increases the number of large follicles 5-7 days after PG treatment. Administration of Neutra-eCG 60 h after PG treatment can partly reverse this trend but has no effect on ovulation rate. The possibility that part of the variability in ovulation rates in this study may have resulted from Neutra-eCG been given prior to or at the LH surge, or from the absence or presence of a dominant follicle at the time of eCG treatment cannot be ruled out.     

The effect of ovarian follicle size on pituitary and ovarian responses to copulation in domesticated South American camelids.

The relation of ovarian follicle size to pituitary and ovarian responses to copulation was studied in domesticated South American camelids (llamas and alpacas). Females from each species were divided into four groups according to follicle size: small (4-5 mm), growing (6-7 mm), mature (8-12 mm), and regressing (10-7 mm). The pituitary response to copulation was determined by analysis of LH and FSH concentrations in plasma. The ovarian response to copulation was determined by ultrasonography and by analysis of estrone sulfate (follicular status) and pregnanediol glucuronide (luteal status) concentrations in urine. Females with small follicles (4-5 mm) released less LH after copulation than did those with larger follicles, and ovulation was not induced. Females with growing and mature follicles (7-12 mm) released LH in response to copulation that was adequate to induce ovulation and to initiate normal luteal activity. While copulation-induced LH release in females with regressing follicles was similar to that released in animals with growing and mature follicles, regressing follicles were luteinized instead of being ovulated. The luteal structure formed as a result of luteinization of follicles had a short life span, i.e., 5.1 days. Copulation-induced LH release was significantly higher in llamas vs. alpacas in animals with mature or regressing follicles, but not in those with small or growing follicles. Urinary estrone sulfate and pregnanediol glucuronide concentrations correlated positively with the presence of follicles and corpora lutea, respectively.     

Relationships between ovarian blood flow and ovarian response to eCG-treatment of dairy cows

The goal of the present study was to investigate ovarian blood flow and ovarian response in cows undergoing a gonadotropin treatment to induce a superovulatory response, using transrectal colour Doppler sonography. Forty-two cows including 19 cross-bred, 14 German Holstein and 9 German Black Pied cows were examined sonographically before hormonal stimulation on Day 10 of the oestrous cycle, three days after administration of eCG (Day 13) and seven days after artificial insemination (Day 7(p.i.)). After each Doppler examination, blood was collected for determination of total oestrogens (E) and progesterone (P4) in peripheral plasma. The blood flow volume (BFV) and pulsatility index (PI), which is a measure for blood flow resistance, were determined in the ovarian artery, and B-mode sonography was used to count dominant follicles and corpora lutea. Important criteria to assess the ovarian response following the hormonal treatment were the number of follicles >5mm in diameter on Day 13 and the number of corpora lutea on Day 7(p.i.) per cow. The number of follicles ranged from 2 to 61 (mean+/-S.E.M.: 17.5+/-1.7) and corpora lutea from 0 to 50 (mean+/-S.E.M.: 17.0+/-1.6). The BFV increased from 28.4 to 45.0 ml/min between Days 10 and 13 and reached a maximum of 108.5 ml/min on Day 7(p.i.) The PI decreased from 6.25 on Day 10 to 4.70 on Day 13 and to 2.10 on Day 7(p.i.) The BFV and PI on Day 13 did not correlate with the number of follicles (P>0.05). However, on Day 7(p.i.) the number of corpora lutea correlated positively with the BFV (r=0.64; P<0.0001), and an inverse relationship was found for the PI (r=-0.51; P=0.0005). There were no correlations (P>0.05) between the BFV and PI on Day 10 and the number of follicles on Day 13 or the number of corpora lutea on Day 7(p.i.) Results of the present study show that in cows, a hormonal treatment to induce a superovulatory response yielded a marked increase in BFV and a marked decrease in PI in the ovarian artery. However, there was no correlation between BFV and PI in the ovarian arteries before hormonal stimulation and the number of follicles and corpora lutea that developed after stimulation. Thus BFV and PI measured in the ovarian arteries have limited diagnostic value to predict the outcome of a gonadotropin treatment.     

Changes in content and phosphorylation of cytosol proteins in luteinizing ovarian follicles and corpora lutea

Cytosol prepared from rat preovulatory ovarian follicles contained several specific substrates which were phosphorylated by [gamma 32P] ATP in the presence of 2 microM cyclic AMP (cAMP) or 780 nM of highly purified catalytic subunit. These substrates were identified as RII, the regulatory subunit of type II cAMP-dependent protein kinase, an Mr = 43,000 protein presumed to be actin, and four other proteins with Mr = 36,500-15,000. A marked decrease in phosphorylation of these proteins was observed within 6-48 h of human chorionic gonadotropin (hCG)-induced ovulation and luteinization in hormonally primed immature rats. The phosphorylation of these proteins was also low in cytosol of corpora lutea isolated on Days 2, 4, 9, 13 and 23 of pregnancy. The decrease in phosphorylation of RII was associated primarily with a decrease in substrate content as measured by photoaffinity labeling and silver staining techniques, and not to a marked increase in phosphoprotein phosphatase and adenosinetriphosphatase (ATPase) activities. Whereas the decreased phosphorylation of other proteins is also presumed to be related to a decrease in their cytosol content, the data do not exclude the possibility that luteal tissue contains a specific phosphoprotein phosphatase which is not present in granulosa or theca cells of preovulatory follicles. We conclude that luteinizing hormone (LH) or hCG, and thereby cAMP itself, induces the rapid loss of specific phosphoproteins which may be involved in regulating cAMP action in granulosa cells.     

Pituitary control of ovarian activity in the lizard, Agama agama

The principal ovarian structures in Agama agama are non-yolky and yolky follicles, corpora lutea and atretic follicles. Hypophysectomy affects all stages of vitellogenesis and follicular atresia results. Atresia involves resorption and digestion of yolk by the granulosa cells, which eventually undergo a fatty degeneration. Immature follicles and corpora lutea are not affected by hypophysectomy.
Injections of 0.2 mg FSH (NIH-FSH-S3 ovine) on alternate days for 14 days not only maintain vitellogenesis in hypophysectomized animals, but also tend to overstimulate the small follicles to synthesize yolk. A similar dose of LH(NIH-LH-SII ovine) fails to maintain vitellogenesis in hypophysectomized animals, while a higher dose of 0.4 mg LH tends to overstimulate the small follicles. An unusual rupture of yolky follicles in FSH injected lizards is reported and a possible development of such follicles into corpora lutea is discussed.     

Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary.

The localization of mRNAs for insulin-like growth factors I (IGF-I) and II (IGF-II) and the type 1 IGF receptor (IGF-1R) in bovine follicles and corpora lutea was determined using in situ hybridization on sectioned ovaries collected from nonpregnant, cyclic Holstein cows in either the follicular (n = 3) or luteal (n = 5) phases of the cycle. Concentrations were measured as absorbance units of individual regions or follicles from autoradiographs. There was intense follicular expression of mRNAs encoding IGF-II and IGF-1R. For mRNA encoding IGF-II, expression was significantly higher in smaller follicles (< 5 mm diameter, P < 0.01) and, in this size range, expression was significantly greater in healthy compared with atretic follicles (P < 0.01). For mRNA encoding IGF-1R, there was no effect of size but concentrations were again significantly greater in healthy compared with atretic follicles of < 5 mm. In medium (5-10 mm) and large (> 10 mm) follicles, there was no effect of health for expression of either IGF-II or IGF-1R. mRNA encoding IGF-II was found exclusively in the theca, whereas mRNA encoding IGF-1R was confined to the granulosa layer. IGF-I expression was not detectable in 83% of the 53 follicles examined. In the remaining 17% of follicles, expression was very low and was unrelated to size or state of atresia. mRNAs encoding IGF-I, -II and IGF-1R were all present in the corpus luteum, whereas only those for IGF-II and IGF-1R were found in ovarian stroma. These data indicate that the insulin-like growth factors play a significant role in follicular and luteal development in the bovine ovary. Locally produced IGF-II is probably an important regulator of follicular growth, whereas most of the IGF-I present in follicular fluid is likely to be derived from the circulation.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.