Short FSH Research Articles

Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe)

The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary–gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33°38′28″S, 70°34′27″W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 17β-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 17β-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.

A Novel Mutation in SOST Gene Causes Sclerosteosis

Background: Sclerostin is a SOST gene product that inhibits osteoblasts activity and prevents excessive bone formation by antagonizing Wnt signaling pathway. Sclerosteosis has been linked to the loss of function mutation in SOST gene. It is a rare autosomal recessive disorder characterized by craniotubular hyperostosis leading to gigantism, cranial nerves entrapment, and fatal cerebellar herniation. Objectives To report a novel mutation of SOST gene in a patient with sclerosteosis. Clinical Case: A 25-year-old female was referred to the endocrine clinic for suspected GH excess. The patient noted the onset of headache, progressive bilateral blurred vision and hearing disturbance, irregular menses, and generalized arthralgia; at the age of 23 years. Subsequently, she observed a progressive increase in the size of shoes and hands, proptosis, and protrusion of the chin. She was the second of seven siblings from non-consanguineous parents with normal antenatal and neonatal history except for syndactyly. All family members were phenotypically normal except for a sister with similar physical appearance who had cranial decompression 20 years back. MRI pituitary was done initially due the suspicion of pituitary adenoma and it revealed an enlarged sella turcica with normal pituitary gland. Surprisingly, the MRI showed diffuse osseous thickening with narrowing of skull base foramina, narrowing of optic and internal auditory canals, secondary compression of cerebral parenchyma and bilateral cerebellar tonsillar herniation. Further image revealed extremely increased bone mass density with Z-score values of +12, generalized increase cortical thickness, vertebral end plates sclerosis, and deformed left index finger. Biochemical and endocrine tests revealed normal GH, IGF -1, TSH, prolactin, short Synacthen test, FSH, LH, estradiol, calcium, phosphorus, PTH and alkaline phosphatase. Due to progressive worsening of vision with compressive optic neuropathy, optic nerve fenestration with decompression hemicraniotomy was performed. Sclerosteosis was suspected due to the predominant craniotubular hyperostosis with syndactyly. There was no definite therapy. Management aimed at relieving symptoms and preventing complications, so she was commenced on calcitriol and prednisolone to suppress the osteoclasts. Genomic sequencing of the SOST was performed. We identified a novel deletion mutation in SOST gene (c.387delG, p.D131fs*) which disrupts the sclerostin function causing sclerosteosis in this patient. Conclusion: We describe a novel mutation in the SOST gene in a patient with sclerosteosis in Saudi Arabia, that has not been previously described. Closing the gap between the genomic knowledge and clinical applications will add the benefit of success in development of targeted therapies in such a fatal disease.

FSH may be a useful tool to allow early diagnosis of Turner syndrome

BackgroundUltrasensitive assays to measure pre-pubertal gonadotropins levels could help identify patients with Turner syndrome (TS) in mid-childhood, but studies in this field are scarce. The aim of this study was to analyze gonadotropins levels in girls with TS throughout childhood.MethodsRetrospective longitudinal study conducted with 15 girls with TS diagnosed with < 5 years whose FSH and LH measures were available since then. Hormones were evaluated in newborn/mini-puberty (< 0.5 years), early childhood (0.5–5 years), mid-childhood (5–10 years) and late childhood/adolescence (> 10 years). In newborn/mini-puberty and late childhood/adolescence pre-pubertal or pubertal gonadotropins were considered normal; in early childhood and mid-childhood concentrations above the pre-pubertal range were considered abnormal.ResultsAbnormally high FSH alone was found in four of five patients in newborn/mini-puberty, 13 of 15 during early childhood and nine of 15 during mid-childhood. In the group of 12 patients in late childhood/adolescence, the three girls with spontaneous puberty had only normal levels; the remaining showed only post-menopausal concentrations. In mid-childhood one patient exhibited only pre-pubertal FSH. Conversely, most LH measurements in early and mid-childhood were normal.ConclusionKaryotyping of girls with short stature and high FSH levels would allow early diagnosis of Turner syndrome in a significant number of patients, particularly when resources for chromosome study of all girls with growth deficiency are limited.

Effect of follicular aging on ATP content and mitochondria distribution in bovine oocytes

Hereford heifers were assigned randomly to three superstimulation groups and given FSH for 4 days in the short FSH group (n = 5) and FSH starvation group, (n = 5) or for 7 days in the long FSH group (n = 4). In vivo oocyte maturation was induced with LH given 12 hours after the last FSH treatment in short and long FSH groups and 84 hours after the last FSH treatment in the FSH starvation group. The ovaries were removed by colpotomy 18 to 20 hours after LH treatment to aspirate cumulus–oocyte complexes (COCs) from follicles 8 mm or greater. Cumulus–oocyte complexes were graded morphologically, and oocytes were processed for either mitochondrial staining or for ATP assay. Collection efficiency was similar among treatment groups, but a greater proportion of COCs were expanded (P < 0.01) and oocyte ATP content of the expanded COC tended to be greater (P < 0.09) in the long FSH group than other two groups. Oocytes in the FSH starvation group had a greater proportion (P = 0.01) of mitochondrial clusters (i.e., fewer scattered individual mitochondria). Individual mitochondria and mitochondrial clusters in oocytes from the long FSH and FSH starvation groups had twice the relative staining intensity (P < 0.01) compared to oocytes from the short FSH group. In summary, the long FSH superstimulation protocol yielded a greater proportion of expanded COCs that had oocytes with a scattered mitochondrial population and a greater ATP content than other two protocols. FSH starvation of 84 hours yielded a high proportion of grade 4 COCs characterized by a greater proportion of mitochondrial clusters within the oocyte.

IVM in need of clear definitions.

Oocyte in vitro maturation (IVM) involves the achievement of the process of oocyte maturation in vitro Clinically, it was introduced several decades ago as a potentially more patient-friendly and less expensive assisted reproductive technology (ART) approach, as immature oocytes collected from mid-antral follicles can be matured in vitro without prior gonadotrophin stimulation. However, IVM oocytes are developmentally less competent compared with oocytes matured in vivo, a fact that has encouraged the use of short FSH priming and/or hCG triggering in IVM cycles. These alternatives have generated much confusion about the definition and clinical outcome of IVM, also among ART specialists, especially because hCG triggering can support maturation in vivo even in follicles of 10-13 mm in diameter. In a recent manuscript, a team of IVM specialists propose that IVM should include any ART approach involving 'the collection of oocyte from small and intermediate sized follicles' even after hCG or GnRH triggering. It is more than predictable that other scientists and clinicians with an interest in IVM will object to such a definition, believing that semantically and operatively IVM should not be associated with pharmacological interventions aimed at promoting or achieving maturation in vivo, although partially.

287 EFFECT OF FOLLICULAR AGING ON THE ATP CONTENT AND DISTRIBUTION OF MITOCHONDRIA IN BOVINE OOCYTES

Our objective was to determine how follicular aging affects the distribution and content of mitochondrial population and ATP in in vivo-matured bovine oocytes. We hypothesised that in vivo-matured bovine oocytes obtained from aged follicles (84 h of gonadotropin starvation) have altered mitochondrial distribution and decreased cytoplasmic ATP content compared to those obtained immediately at the end of a superstimulatory protocol (no starvation). Follicular waves were synchronized by ablation 5 to 8 d after ovulation and a CIDR device was given. Starting on the day of wave emergence (Day 0), short FSH and FSH starvation groups (n = 5 heifers each) were given 8 doses of FSH im over 4 d and the long FSH group (n = 4) was given 14 doses over 7 d. Two doses of PGF were given on Day 4 (short FSH) or Day 7 (FSH starvation and long FSH groups), the CIDR was removed, and LH was given 24 h after second PGF treatment. The ovaries were removed 24 h later by colpotomy and cumulus-oocyte-complexes (COC) were collected from follicles ≥8 mm. Denuded oocytes were either stained with Mitotracker Deep Red FM and imaged by confocal microscopy or processed for ATP assay. Mitochondria numbers were assessed by segmentation of 3D datasets. Proportions of COC within each grade were compared using Fischer's exact test, and ATP and mitochondrial data were compared by analysis of variance. Short and long FSH groups had a greater proportion of Grade 1 expanded COC than the FSH starvation group (P = 0.02). The ATP content of oocytes (from expanded COC) tended to be higher in the long FSH group than short FSH (P = 0.09), and the FSH starvation group was intermediate. The ATP content of oocytes from compact COC did not differ among groups (P = 0.49). The proportion of mitochondrial clusters was highest (P = 0.01) and the proportion of individual mitochondria was lowest (P = 0.01) in the FSH starvation group compared to short and long FSH groups. Mitochondria from the long FSH and FSH starvation groups had twice the relative intensity compared to the short FSH group (P &lt; 0.01). In conclusion, follicular aging (FSH starvation) was associated with a decrease in oocyte morphologic grade and marked clustering of mitochondria, which may be a reflection of oxidative stress and atresia.

Effect of superstimulation protocols on nuclear maturation and distribution of lipid droplets in bovine oocytes.

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n=4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24h after induced luteolysis (penultimate day of FSH) and cumulus-oocyte complexes were collected 24h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P<0.01). On average across all groups, oocytes contained 22pL of lipids (3.3% of ooplasm volume) distributed as 3000 droplets. Average volume of individual lipid droplets was higher in the FSH Starvation (11.5±1.5 10(-3) pL, P=0.03) compared with the Short and Long FSH groups (7.2±0.6 10(-3) and 8.0±0.8 10(-3) pL, respectively). In conclusion, both FSH Starvation and Short FSH treatments yielded a lower proportion of mature oocytes compared with the Long FSH treatment. Furthermore, FSH starvation led to an accumulation of larger lipid droplets in the ooplasm, indicating atresia. Our results indicate that a longer superstimulation period in beef cattle yields higher numbers and better-quality oocytes.

Length of the follicular growing phase and oocyte competence in beef heifers

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were randomly assigned to three groups: short FSH, FSH starvation, and long FSH (N = 8 per group). At 5 to 8 days after ovulation, follicle ablation was performed, and a progesterone-releasing device (CIDR) was placed intravaginally. Short FSH and FSH starvation groups were given eight doses of FSH im at 12-hour intervals, and the long FSH group was given 14 doses. PGF2α was administered twice (12 hours apart) and the CIDR was removed on Day 3 (Day 0 = wave emergence) in the short FSH group, and on Day 6 in the other two groups. Heifers were given LH 24 hours after CIDR removal and cumulus-oocyte complexes (COC) were collected 24 hours later. The COC were matured in vitro for 6 hours and fertilized in vitro; embryos were cultured for 10 days. A greater number of follicles ≥9 mm were detected in the long FSH group than in the FSH starvation and short FSH groups (25.4 ± 5.3, 11.0 ± 2.1, 10.6 ± 2.3, respectively; P < 0.03). A greater proportion of expanded COC were collected from the long FSH than from the FSH starvation group (P < 0.001), and the short FSH group was intermediate (93%, 54%, and 74%, respectively). The FSH starvation group had a greater proportion of poor quality oocytes than the short and long FSH groups (70%, 45%, and 33%, respectively; P < 0.001) and cleavage rate was lower (22%, 54%, and 56%, respectively; P = 0.003). The proportion of oocytes that developed into embryos (morulae and blastocysts on Day 9 after IVF) was also lower in the FSH starvation group than in the short and long FSH groups, (5% vs. 25% and 37%; P = 0.04); the latter two groups did not differ. The long FSH treatment resulted in 2.5 and 3.4 times more transferable embryos per animal (morulae and blastocysts) at Day 9 after IVF than the short FSH and FSH starvation groups (5.6, 2.5, and 1.7 embryos per heifer respectively; P = 0.04). In conclusion, extending the standard superstimulation protocol by 3 days enhanced the ovarian response to FSH treatment, and a period of FSH starvation after superstimulatory treatment compromised oocyte quality and the fertilization process.

313 EFFECT OF DURATION OF THE GROWING PHASE OF OVULATORY FOLLICLES IN SUPERSTIMULATED HEIFERS ON OOCYTE COMPETENCE AFTER IN VITRO FERTILIZATION

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were allocated randomly to short FSH duration (n = 8), FSH starvation (n = 8), or long FSH duration (n = 8) groups. Five to 8 days after ovulation, transvaginal ultrasound-guided follicle ablation was done to synchronize follicle wave emergence, and a progesterone-releasing device (CIDR; Pfizer Animal Health, New York, NY, USA) was placed intravaginally. Short FSH and FSH starvation groups were given 8 doses of FSH (Folltropin-V; Bioniche Animal Health Inc., Belleville, ON, Canada) IM, whereas the long FSH group was given 14 doses of FSH at 12-h intervals, starting from the day of wave emergence (Day 0). Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 3 in the short FSH group and on Day 6 in the other 2 groups. In all heifers, the CIDR was removed at the time of the second PGF treatment; pLH (Lutropin-V; Bioniche Animal Health Inc.) was given IM 24 h after CIDR removal, and cumulus–oocyte complexes (COC) were collected 24 h after pLH treatment. The COC were matured in vitro (6 h) and fertilized (IVF), and the embryos were cultured for 10 days. At 12 h after pLH, the long FSH group had a greater number of ≥9 mm follicles than the FSH starvation and short FSH groups (25.4 ± 5.3 v. 11.0 ± 2.1 and 10.6 ± 2.3, respectively; P &lt; 0.03). The long FSH group also had more expanded COC than the FSH starvation group (P &lt; 0.001), but did not differ from the short FSH group (93, 54, and 74%, respectively). The FSH starvation group had a greater proportion (P &lt; 0.0001) of partially expanded COC (32%) and poor quality oocytes (70%) than did the long (1 and 33%) and short (4 and 45%) FSH groups; oocyte quality did not differ between long and short FSH groups. At 48 h after IVF, the cleavage rate was lower in the FSH starvation group compared with the short and long FSH groups (35, 54, and 56%, respectively; P = 0.003). After 9 days in culture, embryo development (morula + blastocyst) in the FSH starvation group was lower than that in the long FSH group, (18 v. 37%; P = 0.04), but did not differ from that in the short FSH group (25%). After removal of the data of one heifer in the FSH starvation group that produced 52% of total embryos in that group (outlier), the Day 9 blastocyst rate was lower in the FSH starvation group than in the short and long FSH groups (2% v. 14 and 21%, respectively; P = 0.02). In conclusion, extending the standard superstimulation protocol by 3 days enhanced ovarian response to FSH treatment, but did not improve oocyte competence, whereas a period of FSH starvation after FSH treatment compromised oocyte quality and embryo development.

Effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n=14), FSH starvation (n=13) and long FSH (n=13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P=0.001), and ova and/or embryos (P<0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate.

Inhibin secretion in the golden hamster (Mesocricetus auratus) testis during active and inactive states of spermatogenesis induced by the restriction of photoperiod.

Gonadal function in the male golden hamster (Mesocricetus auratus) was investigated during exposure to a short photoperiod condition. Within 3 weeks of exposure to the short photoperiod condition, FSH and testosterone in the plasma significantly decreased, and subsequently immunoreactive (ir)-inhibin significantly decreased. Testicular contents of ir-inhibin and testosterone, and pituitary contents of LH and FSH also significantly decreased by 3 weeks with regression of weight of testes, epididymis and seminal vesicles and sperm head count. Circulating LH varied but not significantly. Thereafter, all reproductive parameters and secretion of LH, FSH, ir-inhibin and testosterone gradually recovered after 17 weeks of exposure even though animals continued to be subjected to the short photoperiod condition. Plasma concentrations of inhibin B and inhibin pro-alphaC were detectable and were significantly decreased after 15 weeks of exposure to the short photoperiod, but their levels were still detectable. Immunopositive reaction of inhibin alpha and betaB subunits was found in Sertoli cells and Leydig cells in the regressed testes of animals subjected to short photoperiod as was also seen in animals before exposure to the short photoperiod. Although the spermatogenic cycle was suppressed like prepubertal animals, the present study showed that the testicular recovery, so-called refractoriness, is functionally different from the developing stage of immature animals, especially with regard to inhibin secretion. The present results showed that changes in FSH preceded changes in inhibin during the regression and recovery phases, indicating that FSH is a major regulatory factor of inhibin secretion in male golden hamsters. The present study also demonstrated that regressed testes still secrete a small amount of bioactive inhibin during exposure to a short-photoperiod condition.

Short time priming of pig cumulus-oocyte complexes with fsh and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50–200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 u M) and FSH (100 U/L) priming CEO could timedependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosisactivating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.

Two-dimensional gel analysis of Sertoli cell protein phosphorylation: effect of short term exposure to follicle-stimulating hormone.

The response of cultured Sertoli cells to short term FSH stimulation was studied to elucidate early events involved in the hormone response of this cell type. The phosphorylation of proteins by [32P]orthophosphate-labeled cells was examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. FSH stimulation resulted in a variety of changes in the phosphoprotein labeling pattern. Within 5 min, unique phosphoproteins appeared in autoradiograms of treated cells. Increased labeling of vimentin was also noted. After 25 min of FSH stimulation, increases and decreases in apparent labeling intensity were evident in additional phosphoproteins that were constitutively labeled in control cultures. Changes in protein patterns in stained gels were also noted after acute hormone treatment. These observations demonstrate that Sertoli cells respond to hormonal stimulation in a detectable manner within 5 min. Cytoskeletal involvement in initial phases of hormone response is indicated. Mechanisms such as increased protein kinase activity, changes in protein kinase substrate affinity, increased protein turnover, or phosphatase activation may all contribute to the early events involved in Sertoli cell hormone response.