F1 Follicles Research Articles

Subdominant hierarchical ovarian follicles are needed for steroidogenesis and ovulation in laying hens (Gallus domesticus)

Ovarian follicle development in avian species is characterized by a strict hierarchical arrangement. The hierarchical follicles secrete progesterone, which induces the LH surge, but the capacity to produce other steroids decreases with development. Our aim was to evaluate the complementary action of subdominant follicles (F4–F6) on ovulation and steroidogenesis of the preovulatory follicles (F1–F3) in domestic laying hens. The first study included four groups: control (C); sham-operated (SO); large hierarchical follicles (LHF) from which F4–F6 follicles were extracted; and subdominant hierarchical follicles (SHF) from which F1–F3 follicles were extracted. Blood samples were collected every 2h from 12h before estimated ovoposition until 2h after ovoposition. Egg laying continued at the same rates in C and SO hens, with normal preovulatory surges of oestradiol, testosterone, progesterone and LH. In contrast, in LHF and SHF groups, ovoposition was blocked; oestradiol concentrations were not affected; but no preovulatory surges of testosterone, progesterone or LH were seen. Further, the testosterone surge was required for the occurrence of progesterone and LH surges. In the second study StAR and steroidogenic enzyme mRNA expression was evaluated within F1–F3 follicles from a LHF group and C-14 and C-8 controls groups, in which follicles were collected 14h and 8h before expected ovoposition, respectively. Extraction of F4–F6 follicles caused a significant reduction in StAR and 3β-HSD expressions within theca, but not in granulosa cells. In conclusion, subdominant hierarchical follicles (F4–F6) are required for the preovulatory release of testosterone, progesterone and LH, which are highly inter-correlated.

Effect of adenovirus-mediated up-regulation of α-enolase gene products on follicle-stimulating hormone receptor mRNA and luteinizing hormone receptor mRNA of granular cells from goose F1 follicles

Enolases are glycolytic enzymes in the glycolytic pathway. In order to evaluate the effect of ENO1 on follicle-stimulating hormone receptor (FSHR) mRNA and luteinizing hormone receptor (LHR) mRNA of primary granular cell from goose F1 follicles, the recombinant plasmid adenovirus carrying ENO1 were constructed and infected the primary culture granular cells. The granular cells were randomly divided into three groups: recombinant adenovirus infected (pAd-CMV-ENO1), empty vector infected (pAd-CMV-Null) and no virus (mock control). The expression levels of FSHR mRNA and LHR mRNA of granular cells were examined by qRT-PCR. The results showed the group pAd-CMV-ENO1 had significantly higher FSHR mRNA expression levels than the other two groups (P < 0.05), but had significantly lower LHR mRNA expression levels than the other two groups (P < 0.05). The results suggested that ENO1 could improve the combination rate between FSH and FSHR to accelerate the proliferation and differentiation and steroidogenesis in poultry gonadal tissues.

Molecular characterization, tissue distribution, and expression of two ovarian Dicer isoforms during follicle development in goose (Anser cygnoides)

Dicer plays a key role in the biogenesis of microRNAs and small interfering RNAs, which control the coordinated expression of multiple of genes during follicle development. In this study, the cDNAs encoding two Dicer isoforms (gDicer-a and gDicer-b, respectively) were isolated and cloned from goose ovary using RT-PCR. This is the first time a new Dicer splice variant has been characterized at the molecular level in vertebrates. Sequence analysis indicated that both of the two isoforms consist of seven conserved functional domains, where gDicer-b lacks a linker sequence between DEAD box and helicase C domain composed of 158 amino acids. Each domain of gDicer-a/gDicer-b showed higher than 89.5% identity to corresponding domain of Dicers from chicken, human, and mouse. The ubiquity of transcripts of gDicer-a/gDicer-b was found in all tested tissues by real time PCR with the pituitary, oviduct, and hypothalamus being the predominant site of expression of gDicer-a. A similar expression profile of the gDicer-a/gDicer-b mRNAs was found during follicle development. The abrupt changes in transcripts of gDicer in 2-4mm, 9-10mm, F5, and F1 follicles support its participation in the process of follicle recruitment, selection, dominance, and ovulation. However, high mRNA levels of gDicer-b and caspase-3 were detectable in atretic and post-ovulatory follicles, where expression of gDicer-a was considerably low. These findings suggest that gDicer is required for follicle development, and structural differences in the helicase domain of two gDicer isoforms might contribute to their different roles in controlling granulosa cell apoptosis.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on secretion of steroids and STAR, HSD3B and CYP19A1 mRNA expression in chicken ovarian follicles

The aim of the study was to investigate the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid hormone secretion by chicken ovarian follicles and mRNA expression of genes involved in steroids synthesis. In the first in vitro experiment, white (WF) and yellowish (YF) follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest yellow preovulatory follicles (F3–F1) were incubated in a medium supplemented with TCDD (0.01–100nM). In the second experiment, they were incubated in a medium with TCDD (10nM), ovine LH (10ng/mL; oLH) or a combination of oLH (10ng/mL) and TCDD (10nM). It was found that TCDD decreased estradiol (E2) secretion by WF and the TL of all preovulatory follicles, testosterone (T) secretion by WF, YF, and the TL of F2 and F1 follicles, and progesterone (P4) secretion by the GL of the preovulatory follicles. It also reduced oLH-stimulated E2 and P4 secretion by all examined follicles and T by WF. Real-time qPCR revealed that TCDD affected basal and oLH-stimulated expression of STAR, HSD3B and CYP19A1 mRNAs in all investigated ovarian follicles. In conclusion, the data obtained indicate that TCDD inhibits sex steroids secretion from chicken ovarian follicles. The effects of TCDD depend on its concentration and the stage of follicle maturation, and are associated with modulation of STAR, HSD3B and CYP19A1 mRNAs expression. These results indicate that the exposure of the laying hen to TCDD by influence of ovarian steroidogenesis may impair the selection of white follicles to preovulatory hierarchy and disturb their growth and preovulatory maturation.

Transcription Factors GATA-4 and GATA-6: Molecular Characterization, Expression Patterns and Possible Functions During Goose (&lt;i&gt;Anser cygnoides&lt;/i&gt;) Follicle Development

The transcription factors GATA-4 and GATA-6, members of the GATA family, play an important role in ovarian cell proliferation, differentiation and apoptosis. In this study, the full-length coding sequences of goose GATA-4 and GATA-6 were cloned and characterized. GATA-4 and GATA-6 consist of 1236 and 1104 nucleotides encoding proteins with 411 and 367 amino acids, respectively. The deduced amino acid sequences of both proteins include two adjacent zinc finger domains with the distinctive form (CVNC-X17-CNAC)-X29-(CANC-X17-CNAC) and share 84.76% identity within this domain. In silico prediction together with matching of the high affinity RRXS(T)Y motif revealed that the GATA-4 protein might be phosphorylated predominantly at S233, but no phosphorylation site was found in the GATA-6 protein. Real-time quantitative PCR analysis showed that GATA-4 and GATA-6 mRNAs were co-expressed in goose follicles, moderately expressed in granulosa cells and weakly expressed in theca cells. The expression level of GATA-4 mRNA in healthy follicles was significantly higher than in atretic follicles or postovulatory follicles (P<0.01), and the expression level of GATA-6 mRNA in healthy follicles was significantly lower than in atretic follicles or postovulatory follicles (P<0.01). The expression level of GATA-4 mRNA in granulosa cells was downregulated during follicle development; the peak of expression occurred in the 8-10 mm follicles, and the lowest expression was in the F1 follicles. GATA-6 was upregulated and reached its peak expression in the F1 follicles. These results indicate that the molecular structural differences in goose GATA-4 and GATA-6 may be related to their different roles during follicle development.

Ultrasonographic Characterization of Follicle Deviation in Follicular Waves with Single Dominant and Codominant Follicles in Dromedary Camels (Camelus dromedarius)

Follicular wave emergence was synchronized by treating camels with GnRH when a dominant follicle (DF) was present in the ovaries. Animals were scanned twice a day from day 0 (day of GnRH treatment) to day 10, to characterize emergence and deviation of follicles during the development of the follicular wave. Follicle deviation in individual animals was determined by graphical method. Single DFs were found in 16, double DFs in 9 and triple DFs in two camels. The incidence of codominant (double and triple DFs) follicles was 41%. The interval from GnRH treatment to wave emergence, wave emergence to deviation, diameter and growth rate of F1 follicle before or after deviation did not differ between the animals with single and double DFs. The size difference between future DF(s) and the largest subordinate follicle (SF) was apparent from the day of wave emergence in single and double DFs. Overall, interval from GnRH treatment to wave emergence and wave emergence to the beginning of follicle deviation was 70.6 ± 1.4 and 58.6 ± 2.7 h, respectively. Mean size of the DF and largest SF at the beginning of deviation was 7.4 ± 0.2 and 6.3 ± 0.1 mm, respectively. In conclusion, the characteristics of follicle deviation are similar between the animals that developed single or double DFs.

Regulation of Granulosa Cell Cocaine and Amphetamine Regulated Transcript (CART) Binding and Effect of CART Signaling Inhibitor on Granulosa Cell Estradiol Production During Dominant Follicle Selection in Cattle1

We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.

Lipopolysaccharide in ovarian follicular fluid influences the steroid production in large follicles of dairy cows

In postpartum dairy cows, various inflammatory diseases depress reproductive performance. Lipopolysaccharide (LPS) derived from infections of the uterus or mammary gland with Gram-negative bacteria was shown to suppress steroid production in the granulosa cells of follicles in vitro. The aim of the study was to investigate the relationship between LPS in ovarian follicular fluid and steroidogenesis by the theca and granulosa cells of the large follicles in vivo. Bovine ovaries were collected from a slaughterhouse, and the largest (F1) and the second largest (F2) follicles were used (>8mm in diameter, n=38). LPS concentration in the follicular fluid was measured using quantitative kinetic assay. Follicular steroidogenesis was evaluated by measuring the estradiol (E2) and progesterone (P4) concentration in follicular fluid and by analysing transcription levels of steroidogenesis-related genes in theca and granulosa cells. LPS concentration detected in follicular fluid ranged from 0.2 to 2.0EU/mL. In follicles with a high level of LPS (>0.5EU/mL, n=15), the concentration of E2 was lower and that of P4 was higher when compared to those in follicles with a low level of LPS (<0.5EU/mL, n=23), which was observed both in F1 and F2 follicles. Furthermore, in follicles with a high level of LPS, transcripts of steroidogenic enzymes such as CYP17 and P450arom were lower. In those follicles, the expression of caspase-3 was high, suggesting an association with follicular atresia. These findings indicate that LPS present in follicular fluid may cause ovarian dysfunction by inhibiting follicular activity.

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.

Increased hepatic yolk precursor synthesis, secretion and facilitated uptake by follicles are involved in the rejuvenation of reproductive performance of molted hens (Gallus gallus domesticus)

Molt, a natural behavior that is initiated at the end of a lay cycle in birds, is implicated in the regression of the reproductive system in birds followed by a rejuvenation of egg-laying potential. The aim of the present study was to evaluate the physiological basis for the apparent rejuvenation of egg production that occurs following molting. Eighty-three-week-old Hy-line hens, were obtained and subjected to forced molting. Blood and tissue samples were obtained at the beginning of molt (at 83weeks of age), during molt (at 85weeks of age) and postmolt (at 89weeks of age). The laying performance, egg quality, blood parameters and gene expression in the liver and the ovary were investigated before, during and after molt. There was an obvious increase in the postmolt laying rate from 70% premolt to 93% postmolt. Eggshell thickness, albumin height, Haugh unit and egg shape index were all significantly improved after molt. The circulating levels of estrogen and progesterone were lower in the postmolt hens, whereas the concentrations of luteinizing hormone and follicle stimulating hormone were not significantly affected by molt. These results indicate that enhanced hepatic yolk precursor synthesis and secretion contribute to increased postmolt laying performance. Molt enhanced the sensitivity of sex hormones in F1 follicles. Augmented gene expression in the ovary was involved in the rejuvenation of the reproductive performance of molted hens. These results suggest that facilitated yolk-precursor uptake by follicles is involved in the rejuvenation of the reproductive performance of molted hens.

The characteristics of oviposition and hormonal and gene regulation of ovarian follicle development in Magang geese

BackgroundEgg laying in Magang geese is characterized by extended interruption between clutches and lowing laying rate. Both the ovarian follicular development and ovulation characteristics, and the associated endocrine and molecular regulatory mechanisms involved are poorly understood, but could be important for guiding development of molecule aided selection of egg laying performances in geese. This study, therefore, recorded egg-laying characteristics of Magang geese, and the endocrine and molecular regulatory mechanisms of ovarian follicular development, maturation, and ovulation in Magang geese.MethodsOviposition, ovarian follicle development, and reproductive hormone and gene expression profiles were observed in a small flock of Magang geese.ResultsGreater than 73% of eggs were laid during the day. The average oviposition interval was 46.8 h (36–55 h). It took approximately 18 days for large white follicles to develop into mature F1 follicles; follicular growth was exponential. LHR expression levels increased from the small to the large mature follicles, but FSHR expression decreased in the granulosa and thecal layers. As the follicles matured, inhibin alpha and inhibin betaA expression increased in the granulosa layer. Activin IR, activin IIRA, activin IIRB, and beta-glycan expressions also increased as the follicles increased in size, but were more abundantly expressed in the thecal than in the granulosa layers. During the oviposition cycle, plasma concentrations of gonadal hormones decreased rapidly, whereas the level of PGFM peaked around ovulation. The profiles of activin, inhibin, follistatin, estradiol, and progesterone leading to ovulation were characterized.ConclusionsThe molecular and endocrine mechanisms that regulate follicular development in Magang geese are similar to those in chickens. Moreover, gonadotropin regulation and interaction between activin, inhibin, and follistatin secretion may govern 3-stage maturation in the final preovulatory follicles in Magang geese. The rapid rebound of post-ovulatory secretions of inhibin and follistatin may inhibit recruitment of new SYF recruitment once a sequence of eggs is started, and may limit the egg clutch size to no more than the number of LYFs present before the first sequence egg.

Expression of CCT6A mRNA in chicken granulosa cells is regulated by progesterone

CCT6A, the zeta subunit of the chaperonin containing TCP1 complex, is the only cytosolic chaperonin in eukaryotes and is estimated to assist in the folding of multiple proteins including actin, tubulin, cyclin E, myosin, transducin and the Von Hippel Lindau tumor suppressor. In this study, we examined the expression of CCT6A and progesterone receptor (PGR) mRNA in various tissues of chickens and the regulation of CCT6A and PGR mRNA in ovarian granulosa cells. Northern blot analysis revealed that CCT6A had one transcript and was highly expressed in the ovary tissues from chickens at both the sexually immature and mature stages. CCT6A mRNA expression was increased maximally from pre-hierarchy follicles to F5 follicles and subsequently declined in pre-ovulatory and post-ovulatory follicles. The expression of PGR mRNA exhibited the similar pattern to CCT6A. In granulosa cells isolated from pre-ovulatory follicles, follicle-stimulating hormone (FSH) inhibited the expression of CCT6A mRNA, whereas progesterone activated CCT6A and suppressed PGR expression in a time-dependent manner. We further investigated the regulation of CCT6A transcription by progesterone by constructing various progressive deletions and mutants and identified the core promoter element of CCT6A and the binding region of progesterone, which is located from −2056 to −2051. Taken together, our results indicate that CCT6A likely plays an important role in follicle growth, and in granulosa cells, progesterone activates CCT6A transcription via a progesterone response element (PRE) located in the distal promoter of CCT6A.

Relationships between the ovarian status and superovulatory responses in dairy cattle

The aim of this study was to determine the influence of follicular profiles over 4 days prior to superovulation on superovulatory responses. Eighty-eight Holstein cows were synchronized by two prostaglandin F2α injections given 11 days apart and conventionally superovulated between days 8 and 12 of the estrous cycle with 400mg Folltropin-V given in decreasing doses over 4 days. Luteolysis was induced by 2 im injections of cloprostenol (2ml) with the sixth and seventh injections of Folltropin-V. The ovaries of all cows were examined by ultrasonography with a real-time linear scanning ultrasound diagnostic system (Ls-300-A: Tokyo Keiki Co., Tokyo, Japan; 7.5MHz Transducer) on days −3, −2, −1, 0 (initiation day of the superovulatory treatment=day 0). Data were analyzed by the GLM procedure of the SAS. Animals with a greater diameter of the largest follicle (F1; 13.4 vs 9.8 and 10.1mm; p<0.007) and with a greater difference in the diameter of the first and second largest follicles (7.6 vs 4.5 and 3.8mm; p<0.001) had the greater superovulatory response and produced the greater number of quality I embryos. In conclusion, the diameter of the F1 and the F1–F2 follicles were higher over a 4-day period prior to superovulation in animals yielding a high than a medium and a low number of quality I and I+II embryos.

Vitamin D Regulates Anti-Mullerian Hormone Expression in Granulosa Cells of the Hen1

Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH is expressed by granulosa cells of adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating follicle-stimulating hormone (FSH) sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small follicles but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSHR) in the granulosa layer, although the mechanism is not known. In this study, we tested whether vitamin D (1,25-dihydroxyvitamin D3) regulates expression of AMH mRNA in granulosa cells of the hen. Granulosa cell layers were removed from follicles 3-5 mm and 6-8 mm in size, dispersed, and cultured for 24 h in Medium 199 + 5% fetal bovine serum (n = 7). The medium was removed and replaced with Medium 199 + 0.1% bovine serum albumin and vitamin D (at doses of 0, 10, and 100 nM) and cultured for 24 h. Cells were harvested and RNA was extracted for use in quantitative PCR. Parallel 96-well plates were set up to examine cell proliferation. AMH and FSHR mRNA expressions were evaluated, and all values were standardized to 18S reactions. There was a significant (P < 0.05) dose-related decrease in the expression of AMH mRNA in granulosa cells of 3- to 5-mm and 6- to 8-mm follicles in response to vitamin D. Additionally, FSHR mRNA and cell proliferation were significantly (P < 0.05) increased by vitamin D in both groups. Western blot analysis for the vitamin D receptor (VDR) showed doublet bands at the expected sizes (58 and 60 kDa) in protein isolated from the chicken granulosa layer. Immunohistochemistry was used to identify VDR within the follicle, and it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer, relative to follicle development, was increased (n = 4; P < 0.05) with follicle development, with greatest expression in the F1 follicle. There was no evidence for expression (mRNA or protein) of the calcium-binding protein, calbindin (CALB1), in the ovary or granulosa layer. Overall, these results suggest that vitamin D regulates AMH expression, and thereby may influence follicle selection in the hen.

Effects of kisspeptin-10 on progesterone secretion in cultured chicken ovarian granulosa cells from preovulatory (F1–F3) follicles

The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P4) was investigated in cultured granulosa cells from F1 to F3 follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000nM concentrations tested, 100nM Kp-10 treated for 24h significantly increased P4 secretion in granulosa cells from F1 to F3 follicles. After 24h and 48h of treatment, 100nM Kp-10 showed a significant increase in P4 secretion, while after 72h of treatment P4 secretion was markedly decreased by Kp-10 compared to the control group (P<0.05). F1 and F2/3 cells treated with 100nM Kp-10 for 24h showed significantly increased viability (P<0.05) and which was in parallel to a marked increase in P4 secretion (P<0.01). Real-time PCR results showed that the gene expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc) and the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) in F1 and F2/3 granulosa cells was significantly up-regulated by 24h–100nM Kp-10 treatment (P<0.05 versus P<0.01, respectively). However, there was no significant difference in StAR, P450scc and 3β-HSD protein content between control and the Kp-10 treated group (P>0.05). These results indicate that Kp-10 stimulates P4 secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3β-HSD gene transcription.

Effect of 3,3′,5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T 3) on progesterone (P 4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3′,5′-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T 2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T 3 or 3,5-T 2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P 4 and cAMP in culture media were determined by RIA. Expression of genes involved in P 4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T 3 or 3,5-T 2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P 4 secretion by preovulatory follicles. The 3,5-T 2 elevated P 4 synthesis by F3, had no effect on F2 follicles, and diminished P 4 production by the GL of F1 follicles. It had no effect on LH-stimulated P 4 production; however, it augmented F-stimulated P 4 production by F2 and F1 follicles. Although T 3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T 2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T 3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T 2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR expression in F2 and F1 ones. The effects of 3,5-T 2 caused elevated basal but diminished LH-stimulated HSD3B mRNA levels. In conclusion, data indicate that both iodothyronines are involved in P 4 production in the GL of chicken preovulatory follicles acting alone and additively with LH. Effects of iodothyronines depend on follicle maturation and are associated with modulation of cAMP synthesis and STAR, CYP11A1, and HSD3B mRNA expression. We suggest that iodothyronines participate in maturation and ovulation of chicken follicles.

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows1

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Structure and Steroidogenic Activity of the Granulosa Layer of F1 Preovulatory Ovarian Follicles of the Hen (Gallus domesticus)

The study was performed to determine the structure and steroidogenic activity of granulosa cells derived from the germinal disc region, proximal region and distal region of the largest preovulatory ovarian follicle (F1) of the hen. The study was carried out on 34 Hy-Line Brown egg-laying hens aged 40 weeks. Morphology of the granulosa cells was studied by histological assessment and scanning electron microscopy. Moreover, the level of P4, histochemical activity of 3beta-HSD and expression of 3beta-HSD gene mRNA in granulosa cells of F1 follicle were determined. The findings indicate that the morphology and steroidogenic activity of the granulosa layer in F1 preovulatory ovarian follicle are associated with the region of the follicle. This is consistent with earlier studies. In the germinal disc region the granulosa cells form a multilayer while in the proximal and distal regions granulosa cells form a single layer. Analysis of P4 concentration revealed that its level in granulosa cells was markedly reduced closer to the germinal disc. Moreover, our study demonstrates for the first time the lower histochemical activity of 3beta-HSD and expression of 3beta-HSD mRNA in granulosa cells from the germinal disc region compared with the proximal and distal region.

Increased iNOS gene expression in the granulosa layer of F1 follicle of over-fed and under-fed broiler breeder hens

To clarify the effects of high (20 and 40% more than normal) and low (20% less than normal) daily feed allowance on egg and body parameters, ovarian morphology and plasma glucose, cholesterol, triacylglycerol, leptin-like hormone, nitrite/nitrate and gene expression of inducible nitric oxide synthase (iNOS) in the granulosa layer of F1 follicle, broiler breeder hens (30-week-old) were fed for 30 days. Egg and body parameters significantly changed between treatments (p<0.05). Effect of different level of feed intake on ovarian morphology parameters was significant (p<0.05), except for white follicles. After 30 days of experiment, plasma nitrite/nitrate (as a index of plasma nitric oxide) and leptin-like hormone increased in FI+20% and FI+40% groups as compared to controls (p<0.05). Plasma level of leptin-like hormone also significantly (p<0.05) increased in FI+40% group. The relative amount of iNOS mRNA expression in the granulosa layer of F1 follicles was significantly higher in FI-20% and FI+40% groups than in control group only after 4 weeks of experiment. The amount of these elevations in the FI-20% and FI+40% groups were 32.4% and 60.9% respectively. It was concluded that iNOS gene is normally expressed in follicular granulosa cells of F1 follicle of broiler breeder hens 2-4 hours before ovulation. However, over- and underfeeding of hens increased iNOS expression in F1 follicle, which may be one of the atresia-inducing factors in hierarchical follicles as shown by the significant (p<0.05) increase of shrunken follicles in the ovary of over- and under-fed broiler breeder hens after 30 days of feeding.

Age-dependent role of steroids in the regulation of growth of the hen follicular wall

BackgroundThe ovaries are the primary targets of senescence effects in mammalian and avian species. In the present study, relationships between reproductive aging, sex steroids and the growth pattern of the pre-ovulatory follicle wall were investigated using young hens with long clutch (YLC), old hens with long clutch (OLC), old hens with short clutch (OSC), and old hens with interrupted long clutch (OILC).MethodsExperiment 1: Hens were sacrificed 1.5 and 14.5 h after ovulation. Experiment 2: YLC and OILC hens were sacrificed 3.5 h after treatments with LH and/or aminoglutethimide (AG), an inhibitor of steroid synthesis. Volumes of pre-ovulatory follicles (F1-F5) and plasma concentrations of ovarian steroids were determined. Experiment 3: Granulosa and theca cells from F3 follicles of OSC and/or YLC hens were exposed in vitro to estradiol-17beta (E2), testosterone (T) and LH and the proliferative activity of the cells was examined using CellTiter 96 Aqueous One Solution Assay.ResultsIn YLC and OLC groups, the total volume of F1-F5 follicles rose between 1.5 and 14.5 h after ovulation (P < 0.01), negatively correlating with the plasma level of E2 (P < 0.01). There was no growth of pre-ovulatory follicles in the middle of the ovulatory cycle in the OSC group, with a positive correlation being present between E2 and the follicular volume (P < 0.05). In young hens, AG caused a rise in the total follicular volume. This rise was associated with a fall in E2 (r = -0.54, P < 0.05). E2 enhanced proliferation of granulosa cells from YLC and OSC groups. The proliferative activity of granulosa and theca cells of YLC hens depended on the interaction between T and LH (P < 0.01).ConclusionsThese data indicate for the first time that the growth pattern of pre-ovulatory follicles during the ovulatory cycle changes in the course of reproductive aging. E2 seems to play a dual role in this adjustment; it stimulates the growth of the follicular wall in reproductive aged hens, whereas it may inhibit this process in young birds. T and LH are apparently involved in the growth regulation during the pre-ovulatory surge in young hens.