Rabbit Ovarian Follicles Research Articles

Differential expression of FSHR and LHR genes and proteins during development of rabbit ovarian follicles.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

Effect of Moringa oleifera (Lam) Leaf Extract on Mating Readiness and Development of Rabbit Ovarian Follicles

Moringa oleifera (Lam) is feed rich nutrients and bioactive compaunds including phytosterogen types namely phytosterols and isoflavones. The purpose of thid study was to test moringa leaf extracts with diferent solvents against mating readiness and ovarian foliccle development in rabbit does . Three moringa leaf extracts namely ethanol coarse extract (EKE) ethanol fraction extract (EFE) and hexan fraction extract (EFH) were used in this study. Seven treatments were tried on 28 rabbit does in the extracts in pellet feed at a dose of 0 % (without extract), EFH 0.13%, EFH 0.26%, EFE 0.37, EFE 0.74%, EKE 0.54% and EKE 1.08%. Each treatment is repeated on 4 tails rabbit does. The variables in this study were mating readiness and histology of ovarian follicle development. The results showed that the treatment of EFH administration of 0.26% caused 100% mating readiness and 75% pregnancy rate. The addition of EFH, EFE and EKE treatment led to a decrease in follicle development in the ovaries of lactation rabbit mothers on day 28. From this study it was concluded that the administration of moringa leaf extract increases blood estradiol, resulting in passionate livestock but has the effect of lowering follicle development in the ovaries of rabbits.

Effect of Oxytocin, IBMX and dbcAMP on Rabbit Ovarian Follicles

Oxytocin (OT) and protein kinase A (PKA), a possible intracellular mediator of hormone action in the ovary, can be potent activators of ovarian functions and fertility. Nevertheless, action of OT on ovarian follicle atresia has not been studied yet. Only single administration of PKA activators [3-isobutyl-1-methyl-xanthine (IBMX) and dibutyryl cyclic adenosine monophosphate (dbcAMP)] on ovarian follicle atresia was studied previously. The aim of this study was to examine the effect of OT (single treatment per one reproductive cycle, multiple treatments for three cycles), IBMX and dbcAMP (multiple treatments) on folliculogenesis and follicular atresia in rabbit. The ovarian cycle in control females was induced only by gonadotropins. Experimental females received co-administration of gonadotropins with OT, IBMX or dbcAMP (at 50μg/female). All females were artificially inseminated. Single-treated females were euthanized after 18-19h. Multiple-treated females were euthanized after the third reproductive cycle. Histological sections of the ovaries were prepared and evaluated by a light microscopy. The follicles were divided into four classes according to the structure of granulosa and theca cells as follows: none or small atresia, cystic atresia, obliterative atresia and atresia associated with luteinization. The ovaries from the control and experimental females, treated during one reproductive cycle or three cycles, were compared. Single OT co-administration increased proportion of follicles with atresia associated with luteinization, but not other types of atresia. No influence of multiple OT co-administration on follicular atresia was recorded. Multiple IBMX and dbcAMP co-administration decreased the proportion of atretic follicles and increased the proportion of healthy follicles without atresia.

Generation of Embryonic Stem Cell Lines from Immature Rabbit Ovarian Follicles

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

The camp analogue, dbcAMP can stimulate rabbit reproductive functions: I. Effect on ovarian folliculogenesis, ovulation and embryo production

The aim of our study was to examine the influence of administration of N6,2’-dibutyryladenosine 3’5’-cyclic monophosphate (dbcAMP), a cAMP agonist, on ovarian folliculogenesis and atresia, as well as on reproductive efficiency in rabbits, whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by 20 IU/kg PMSG followed by 35 IU/kg hCG administration. Experimental animals received PMSG and hCG together with dbcAMP (at 5, 25 or 50 μg/animal). After ovulation and insemination, the animals were sacrificed. Ovaries were weighted, histological sections of ovaries were prepared, and the presence of ovulated and not ovulated follicles and different stages of atresia was evaluated by light microscopy. The eggs were flushed from the oviducts after insemination and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived zygotes and embryos reaching hatched blastocyst stage were determined. Administration of dbcAMP (at doses 25 or 50 μg/animal, but not at 5 μg/animal) was able to increase the proportion of follicles with cystic and luteinization-related atresia. Furthermore, dbcAMP (50 μg/animal, but not lower doses) increased the ovarian mass, number of Corpora lutea, number of harvested oocytes, zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that dbcAMP can stimulate rabbit ovarian follicle atresia, ovulation, oocyte, zygote and embryo yield and development. Furthermore, they confirm in the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of dbcAMP in improving animal reproduction and fertility.

Phosphodiesterase Inhibitor 3-Isobutyl-1-Methyl-Xanthine Affects Ovarian Morphology and Stimulates Reproduction in Rabbits

The aim of our study is to examine the influence of administration of 3-isobutyl-l-methyl-xanthine (IBMX), inhibitor of cAMP and cGMP phosphodiesterases on ovarian functions (folliculogenesis, atresia and luteogenesis), as well as on some reproductive parameters in rabbits whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by PMSG followed by hCG administration. Experimental animals received 20IU/kg PMSG and 35IU/kg hCG together with IBMX (at 5, 25 or 50 μg/animal). After ovulation and mating, the animals were sacrificed. Histological slides of ovaries were prepared, and the presence of follicles and different stages of luteinisation and atresia were evaluated by light microscopy. The pronuclear stage eggs were flushed out from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived embryos reaching blastocyst stage were determined. Administration of IBMX was able to increase the proportion of luteinised follicles. Furthermore, IBMX treatment promoted occurrence of atresia in the remaining follicles after the gonadotropin treatment. Finally, IBMX increased the number of Corpora lutea, number of harvested zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that IBMX can enhance the stimulatory effect of gonadotropins on the rabbit ovarian follicle luteinisation, atresia, ovulation, zygote and embryo yield and development. Furthermore, they confirm the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of IBMX in improvement of farm animal reproduction and fertility.

Developmental Expression of the Rabbit 55-kDa Zona Pellucida Protein and Messenger RNA in Ovarian Follicles

The events associated with the early stages of oocyte activation and granulosa cell (GC) differentiation during ovarian follicular development have been investigated using immunological and cDNA probes. Because zona pellucida (ZP) proteins provide excellent markers for oocyte activation during early stages of ovarian development, a cDNA encoding a full-length ZP protein with calculated molecular weight of 57,185 (rc55) and a specific antibody to the corresponding native rabbit ZP protein (R55) were developed. Immunohistochemical analysis of ovaries demonstrated that R55 was localized in follicles of animals 2 weeks and older. R55 was localized in the oocytes of primordial follicles and the oocytes and GCs of primary follicles but was undetectable in the GCs of large antral follicles. GCs were isolated from 6-week-old rabbit ovaries and cultured in defined media to demonstrate expression of rabbit ZP proteins by GCs in vitro. Rabbit ZP proteins were localized in the cytoplasm of cultured GCs if these cells were treated with monensin, a compound that blocks glycoprotein secretion. Northern blot analysis with the cDNA probe rc55 revealed that cultured GCs contained a mRNA for R55 and that there is stage-specific expression of the R55 mRNA in developing rabbit ovaries. Immunoblot analysis demonstrated that R55 is detectable at 4 weeks and accumulates in 6-, 8-, and 12-week ovaries which contain developing primary and secondary follicles. Collectively, these data illustrate developmental expression of R55 in oocytes and GCs of rabbit ovarian follicles.

Luteinization-associated changes in protein stability of the regulatory subunit of the type I cAMP-dependent protein kinase.

The purpose of this study was to determine how RI alpha, the R subunit of the type I cAMP-dependent protein kinase, is regulated in rabbit ovarian follicles in response to the preovulatory luteinizing hormone surge. When soluble extracts from rabbit preovulatory follicles and 7-day-old corpora lutea were photoaffinity-labeled with 8-N3-[32P]cAMP, 3-fold more RI alpha was detected in corpora lutea than in follicles. Based on DEAE-cellulose chromatography, both type I holoenzyme and free RI alpha increased during luteinization. Western blot analysis of soluble extracts obtained from follicles and corpora lutea at various time points after human chorionic gonadotropin (hCG) injection revealed a 6-10-fold increase in RI alpha protein by 5 h after hCG injection. However, based on Northern blot analysis and solution hybridization/RNase protection assays, this increase in RI alpha protein was not due to an increase in RI alpha mRNA. These results suggested that RI alpha subunit levels were post-transcriptionally regulated. Half-life determinations indicated a 2.1-fold increase in the stability of RI alpha when follicles were incubated in the presence of hCG. The effect of hCG on the stability of RI alpha could also be mimicked by forskolin, thus suggesting that a rise in cAMP levels in follicles during the luteinizing hormone surge plays a role in RI alpha subunit stability. We conclude that RI alpha protein is stabilized in follicles by hCG treatment and the consequent rise in follicular cAMP levels.

Direct effect of gonadotropin-releasing hormone agonists on the rabbit ovarian follicle

To determine if gonadotropin-releasing hormone agonist (GnRH-a)-induced oocyte maturation and degeneration can be attributed to the direct actions on the follicle. Mature rabbit follicle culture. The mature follicles were cultured with human chorionic gonadotropin (hCG) (100 ng/mL), buserelin acetate (10(-9) to 10(-6) M), leuprolide acetate (10(-9) to 10(-6) M), or buserelin acetate (10(-7) M) with a GnRH antagonist (10(-8) to 10(-6) M) for 14 hours. The percentage of oocytes achieving germinal vesicle breakdown, the oocyte degeneration rate, prostaglandins (PG) production by mature follicles, and the frequency of fertilization and embryonic development. Gonadotropin-releasing hormone agonist induced the meiotic maturation of follicle-enclosed oocytes in a dose-dependent manner while concomitantly increasing oocyte degeneration. The simultaneous addition of GnRH antagonist inhibited significantly GnRH-a-induced oocyte maturation and PG production by the mature follicles. Furthermore, a GnRH antagonist reversed the oocyte degeneration rate that had been increased by GnRH-a. The rates of normal fertilization and early embryonic development were significantly reduced in the oocytes matured by GnRH-a as compared with those matured by hCG. Gonadotropin-releasing hormone agonist acts directly on mature rabbit follicles to trigger the oocytes to undergo meiotic maturation, but oocytes matured in vitro by GnRH-a are not necessarily cytoplasmically mature.

Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles.

Cycloheximide (5 mg/kg, i.v.) significantly inhibited ovulation in the rabbit when it was administered as early as 20 h before the ovulation process was initiated by hCG, and as late as 1 h after hCG. The ovulation rate was significantly reduced, but follicular biosynthesis of prostaglandins E and F was only partly inhibited. The biosynthesis of progesterone and oestradiol in follicles during the early stages of the ovulation process was also inhibited. Cycloheximide may therefore inhibit ovulation by a mechanism which is different from the action of indomethacin, and this mechanism may involve the suppression of ovarian steroidogenesis.

Effect of time and dose of indomethacin on follicular prostaglandins and ovulation in the rabbit.

This study determined specifically when ovarian prostaglandins (PGs) increase during ovulation and how effectively different doses of indomethacin inhibit PGs and ovulation. Rabbit ovarian follicles were removed at hourly intervals after stimulating the animals with hCG (50 IU/kg). The follicles were homogenized in 0.1 M acetate buffer (pH 4.5), and the PGE and PGF in the extracts were measured by RIA. Ovulation rates were determined by calculating the percentage of mature follicles that ruptured after stimulation by hCG. Before hCG, the normal levels of PGE and PGF were 111.1 +/- 14.7 and 51.0 +/- 6.6 pg/mg follicle, respectively. By 2 h after hCG treatment, PGE and PGF both increased to 162.6 +/- 17.0 and 80.6 +/- 13.3 pg/mg follicle, respectively. Approximately 5 h later, there was a second, sharper increase in both PGs which peaked at 652.6 +/- 63.5 and 345.4 +/- 32.3 pg/mg follicle, respectively, 10 h after hCG treatment, i.e. at the expected time of ovulation. We found that regardless of whether indomethacin was given early or late during the ovulation process, this agent significantly reduced follicular PG production within 5 min after its administration. For example, only 5 min after 10 mg/kg indomethacin were administered 8 h after hCG, PGE and PGF dropped from 317.7 +/- 50.0 and 125.0 +/- 10.5 pg/mg follicle to 93.3 +/- 17.4 and 49.3 +/- 10.5 pg/mg follicle, respectively. Unexpectedly, when graded doses of indomethacin were administered either 1 or 8 h after hCG, there was not a statistically significant correlation between follicular PG levels and ovulation rate. For example, when doses of 1.25, 2.5, 5.0, and 10.0 mg/kg indomethacin were given 8 h after hCG, the PGE and PGF levels at the expected time of ovulation (i.e. 2 h later) were always equal to or less than the PG levels in follicles that had not been stimulated by hCG, yet ovulation proceeded at rates of 46.7 +/- 12.3%, 30 +/- 7.7%, 14.3 +/- 9.3%, and 0%, respectively. Therefore, the results raise questions about the specific role of PGs in the ovulation process.

Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.

Cytochemical localization of acid phosphatase in the rabbit ovarian follicle.

The different cell layers in the follicle wall in rabbit ovaries were examined for acid phosphatase activity during the ovulatory process. The tissue was taken before the process began and at 5, 7, 9, 10 and 11hr after it was induced by human chorionic gonadotropin. The ovaries were perfused with a glutaraldehyde fixative, cut with a microslicer, incubated for 60min at 37°C in Gomori's acid phosphatase medium, and then routinely prepared for light and electron microscopy. Control tissue, which was incubated with the acid phosphatase inhibitor, sodium fluoride, did not reveal any enzyme activity. The results show acid phosphatase reaction product in all the major cell types in the follicle wall. The enzyme is located mainly in lysosomes and Golgi apparatus. It is not present in the electron-dense granules which are characteristic of the cytoplasm of surface epithelial cells. The acid phosphatase in the thecal layers of the follicle appears to be localized, not only in lysosomes and Golgi apparatus, but also in the multivesicular structures which are present in the thecal fibroblasts of this tissue. Collectively, the data indicate that acid phosphatase and other lysosomal enzymes may contribute, at least in part, to the hydrolytic degradation of follicular connective tissue during ovulation.

Effects of pregnant mare's serum gonadotrophin administered in vivo on steroid accumulation by isolated rabbit ovarian follicles.

Various doses (5, 50 and 100 IU) of pregnant mare's serum gonadotrophin (PMS) were administered to sexually mature female rabbits and steroids measured in follicular fluid, ovarian pieces and in incubation media of isolated follicles treated with ovine luteinizing hormone (LH). Follicular fluid progesterone, oestradiol and androgen were increased after PMS injection. Ovarian progesterone content was increased by all doses of PMS, and androgen and oestradiol content only by 50 and 100 IU PMS. Medium sized follicles from rabbits treated with 50 and 100 IU PMS accumulated more progesterone than all follicles from rabbits treated with saline or 5 IU PMS. Androgen accumulation by small follicles from 50 and 100 IU dose rabbits was higher than that by follicles from other treatment groups. Oestradiol accumulation mimicked androgen accumulation. The addition of LH to the medium stimulated the accumulation of progesterone and androgen by all follicles. LH caused an increase in oestradiol accumulation by medium follicles from saline and low dose PMS treated animals. No effect of LH on oestrogen accumulation was seen with large or medium follicles from animals treated with 100 IU PMS or 50 IU PMS for 2 or 3 days. These results suggest that priming of small follicles with PMS can increase their ability to produce androgen which is probably aromatized to oestrogen.

Ultrastructure of ovarian follicles in in vitro perfused rabbit ovaries: response to human chorionic gonadotropin and comparison with in vivo observations

Ovulation may be achieved and studied in an isolated perfused rabbit ovary upon inclusion of human chorionic gonadotropin (hCG) in the perfusion fluid. The ultrastructural features of the rabbit ovarian follicle prior to ovulation in vitro were compared with those in vivo. The perifollicular vasculature was also examined in in vitro perfused rabbit ovaries during the preovulatory interval. Granulosa cells of the preovulatory follicle share many ultrastructural features in vivo and in vitro; however, only small amounts of smooth endoplasmic reticulum (sER) were observed in granulosa cells in vitro after hCG. Ovulation after hCG in the in vitro preparation tends to occur earlier (6 hours) than in vivo (12 hours). Thus, there may be insufficient time and/or gonadotropin exposure to permit full functional development of granulosa cells, as reflected by reduced amounts of sER. Degradation of collagen fibrils was less prominent in the theca externa and tunica albuginea in vitro than in in vivo. Perifollicular capillaries became dilated after hCG, but interendothelial gaps were not observed. Disappearance of surface epithelium in the apex of follicles was similar in vitro and in vivo.

Effects of in vivo administration of steroids on release of 3H 2O from [lβ,2β- 3H] and [1- 3H]androstenedione by isolated rabbit ovarian follicles

In vivo administration of progesterone and testosterone propionate to immature rabbits for 3 days resulted in a decreased ability of isolated ovarian follicles to generate 3H 2O from [1β,2β- 3H]androstenedione. By contrast, follicles isolated from rabbits treated with estradiol benzoate generated 3 times more 3H 2O from the same substrate. Similar results were obtained when follicles were incubated with [1- 3H]androstenedione. A similar pattern of differences was found when follicles from each group were analysed for estrogen content and when incubated with [4- 14C]androstenedione and 14C-labelled estrogens isolated. These data suggest that estrogen may be a stimulant for its own production by rabbit ovarian follicles.

Role of the extra-follicular compartment in the ovulation of isolated rabbit ovarian follicles.

Ovulation was never observed, even in the presence of gonadotrophins, when preovulatory follicles isolated from oestrous does were cultured in vitro (n = 112). If the follicles were co-cultured with pieces of ovarian tissue and gonadotrophins, then 50% ovulated (P less than 0.01). Attempts to replace the ovarian tissue by various steroid hormones or prostaglandins did not result in follicular rupture except when PGE-2 was used (23.8%, P less than 0.05). We suggest that gonadotrophins have an immediate (less than 1 h) action upon the follicle and ovarian interstitial tissue simultaneously. The response of the ovarian tissue to gonadotrophins is essential for eventual follicular rupture and involves PGE-2.

In-vitro ovulation of rabbit ovarian follicles isolated after the endogenous gonadotrophin surge.

Follicular rupture was never observed when preovulatory rabbit follicles were isolated from the ovary before the ovulatory gonadotrophin discharge and placed in hormone-free culture for 10-14 h (n = 48). However, if the follicles were taken 1 h post coitum and cultured under the same conditions, 11/24 (45.8%) ruptured spontaneously. Follicular rupture under these conditions appeared to be chronologically and histologically comparable to that of ovulation in vivo. Culture in the presence of progesterone increased the proportion of follicles ovulating in vitro (17/23 = 73.9%; P less than 0.05). Inhibition in vitro of the synthesis of steroids or prostaglandins suppressed ovulation, although meiotic maturation of the oocyte did take place. These findings indicate that the follicle constitutes an independent entity within the ovary from 1 h post coitum.

In vitro effects of sex steroids on LH-stimulated steroid accumulation by isolated rabbit ovarian follicles

The in vitro effects of progesterone, testosterone and estradiol-17β on steroid accumulation by isolated rabbit follicles were examined. Progesterone had no effect on LH-stimulated androgen accumulation but inhibited LH-stimulated estrogen accumulation at 10 −7 M and 10 −6 M. Testosterone at 10 −5 M but not at 10 −6 M or 10 −7 M, inhibited LH-stimulated progesterone accumulation. LH-stimulated estrogen accumulation was inhibited at all dose levels of testosterone. Estradiol (10 −5 M) inhibited LH-stimulated androgen accumulation and had no effects on progesterone accumulation. It is concluded that the steroidal milieu of follicles can influence their response to LH.

Acute effects of LH on incorporation of [ 35S]-methionine into proteins by isolated rabbit ovarian follicles

The incorporation of [ 35S]-methionine into follicular protein was investigated during 5 min intervals up to 20 min, and correlated with testosterone production. At 5 and 20 min after stimulation with luteinizing hormone (LH) there was no difference in the [ 35S]-labelled proteins from the controls. At 10 and 15 min, LH seemed to decrease the intensity of a protein band with a molecular weight of approximately 25,000 daltons. There was no significant difference in the amount of TCA precipitable radioactivity between control and LH treated follicles. Testosterone production increased within 10 min of stimulation with LH. Under the conditions employed these data suggest that repression of an inhibitor protein may be required for testosterone production by isolated rabbit ovarian follicles.