FSH bioactivity in commercial preparations of gonadotropins

During the past 2 decades, commercial preparations of FSH have been extensively used to superovulate cattle. The problems that have been encountered in superovulation of cattle include high variability in the ovulation rate and subsequent yield of viable embryos. The lack of predictability in superovulatory trials has been attributed to difficulties in standardizing the potency of commercial FSH preparations. Traditionally, FSH potency has been tested in bioassays that utilize specific responses in whole animals or primary cell cultures. Whole animal bioassays lack sensitivity, while primary cell culture bioassays, which use fresh cells, have inherent variability within each preparation. An FSH bioassay that employed a stable chimeric cell line expressing the human FSH-R was used to provide an accurate measurement of FSH bioactivity. The hormonal potency of 2 commercial preparations of FSH used to superovulate cattle was determined using FSH immuno- and bioassays. Commercial FSH preparations differed in potency. One commercial product, prepared in 4 different years, showed no difference in the immunoactive levels of FSH. In the same product stored under identical conditions, FSH bioactivity varied from year to year. There was variability in FSH bioactivity both between and within commercial products. The lack of correlation between bioactivity and immunoactivity of commercial FSH preparations may explain, in part, the variability observed in superovulation of cattle.     

Expression of FSH and its co-localization with FSH receptor and GnRH receptor in rat cerebellar cortex

The expression of follicle-stimulating hormone (FSH) and its receptor in extrapituitary and non-HPG axis tissues has been demonstrated and their non-reproductive functions in these tissues have been found. However, there have been no reports concerning the expression and function of FSH and its receptor in the cerebellum. In our study, immunofluorescence staining and in situ hybridization were used to detect the expression of FSH, double-labeled immunofluorescence staining was used to detect co-localization of FSH and its receptor and co-localization of FSH and gonadotropin-releasing hormone (GnRH) receptor in the rat cerebellar cortex. Results showed that some cells of the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex showed both FSH immunoreactivity and FSH mRNA positive signals; not only for FSH and FSH receptor, but also for FSH and GnRH receptor co-localized in some cells throughout the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex. These suggested that rat cerebellum could express FSH; cerebellum is a target tissue of FSH; FSH may exert certain functions through FSH receptor in a paracrine or autocrine manner; GnRH may regulate FSH positive cells through GnRH receptor in the cerebellum. Our study provides morphological evidence for further functional research on FSH and related hormones in the cerebellum.     

Studies on expression of FSH and its anti-apoptotic effects on ischemia injury in rat spinal cord

Studies indicated that many tissues could express FSH. New functions of FSH have been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat spinal cord. Double-labeled immunofluorescence stain and in situ hybridization were used to study the co-localization of FSH with its receptor and co-localization of FSH with GnRH receptor in rat spinal cord. Spinal cord ischemia injury models were built, TUNEL stain and Fas immunostaining were made to observe the anti-apoptotic effects of FSH to neurons induced by spinal cord ischemia injury. The results found that some neurons and glias of rat spinal cord showed both FSH immunoreactivity and FSH mRNA positive signals; not only FSH and its receptor but also FSH and GnRH receptor co-located in cells of both gray matter and white matter; treatment with certain concentration of FSH before ischemia–reperfusion injury, less TUNEL positive cells and Fas positive cells were found in motor neurons of ventral gray matter in FSH experiment group than that in control group. These suggested that rat spinal cord could express FSH, it is also a target organ of FSH; FSH might exert functions through its receptor by paracrine or autocrine effects; GnRH in spinal cord might regulate FSH positive neurons through GnRH receptor; FSH might inhibit ischemia induced neuron apoptosis by down-regulating Fas expression in spinal cord.     

A study on co-localization of FSH and its receptor in rat hippocampus

It has been known that GnRH, LH and their receptors exist in hippocampal neurons. However, whether FSH and its receptor also exist in hippocampal neurons remained unknown yet. In situ hybridization, double-labeled immunofluorescence stain and double-labeled immunohistochemistry stain in adjacent sections were used in our research to study the distribution, co-localization of FSH and its receptor and co-localization of FSH and GnRH receptor in rat hippocampus. The result found that pyramidal neurons from CA1 to CA4 region and granule neurons in dentate gyrus could express FSH and its receptor, majority of hippocampal neurons co-expressed FSH and its receptor, FSH and GnRH receptor. These suggested that hippocampal neurons not only express FSH but also act as FSH target cells. FSH may regulate the function of hippocampal neurons by ways of paracrine or autocrine. At the same time, GnRH may regulate the function of FSH neuron in hippocampus through GnRH receptor.     

RU486 blocks the secondary surge of follicle-stimulating hormone in the rat without blocking the drop in serum inhibin.

The stimulatory effect of progesterone on the release of the primary surges of serum LH and FSH is well characterized. Less is known about the relationship of progesterone to the secondary FSH surge. We used the antiprogesterone, RU486, to block the action of progesterone and studied the effects on the primary surges of LH and FSH, and especially on the secondary surge of FSH. Proestrous rats were treated with RU486 at 1,200 h. Rats were killed on proestrus and estrus, and serum levels of LH, FSH, and inhibin were measured. In all RU486-treated rats, the primary surges of LH and FSH were significantly attenuated, and the secondary FSH surge was completely abolished, despite a drop in inhibin levels following the primary surges. A high replacement dose of progesterone, delivered immediately after RU486 treatment, did not restore the primary surges of LH and FSH, or the secondary surge of FSH. These data suggest that other factors in addition to a drop in inhibin are responsible for producing the secondary FSH surge.     

The effect of follicle-stimulating hormone (FSH) on the expression of FSH receptor in cultured rat granulosa cells

The acquisition of FSH receptors during folliculogenesis is believed to be a key event in the subsequent development of the follicle. The regulation by FSH of FSH receptor expression and function were further studied using cultured granulosa cells of diethylstilbestrol (DES)-primed immature rats. Incubation of rat granulosa cells with FSH led to a reduction in FSH receptor levels for a short time (6 h), followed by an increase in FSH receptor levels that reached maximum of around 150% of the initial level within 3 days after the addition of FSH. FSH stimulation caused a reduced cAMP response to subsequent FSH treatment and a time course experiment demonstrated that this response was detectable within 30 min of exposure to FSH and reached a plateau after 4 h to 24 h. The recovery of FSH responsiveness in cAMP production of granulosa cells was seen after 48 h of FSH-free interval. Treatment with forskolin (FSK) enhanced the effect of subsequent FSH on the production of intracellular cAMP. Treatment with PMA did not affect the response to subsequent FSH treatment. These data showed that the FSH is essential for the suppression of the FSH receptor function in the adenylyl cyclase pathway. Desensitization of cellular response to continuous agonist stimulation may occur because of changes in the numbers of FSH receptor, as well as changes in the functional properties of the effector system.     

Sexual maturation in female rats: time-related changes in the isoelectric focusing pattern of anterior pituitary follicle-stimulating hormone

Anterior pituitary glands (AP) were obtained from female rats at 5, 15, 18, 21 and 29 days of age, at the time of vaginal opening (VO) and during adulthood on proestrus. The multiple species of follicle-stimulating hormone (FSH) present within the AP were separated by the technique of polyacrylamide gel isoelectric focusing (PAG-IEF) and measured with the NIAMDD rat FSH radioimmunoassay kit. AP's obtained from immature female rats prior to VO contained elevated levels of total FSH as well as all of the species of AP FSH observed in adult rats (and hamsters). However, the majority of the FSH immunoactivity migrated to the most acidic portion of the gel (isoelectric point [pI] value=4.2-3.8). At the time of VO and during adulthood, a decrease in total AP FSH was observed. In addition, a shift in the relative proportions of certain FSH species occurred. The AP's of adult animals contained relatively greater amounts of more basic (pI values 6.0-5.0) forms of FSH compared with immature animals. When each of the AP FSH species isolated from adult animals was tested in a radioligand receptor assay, the most acidic (pI=4.2-3.8) failed to interact with the receptor preparation, while those with pI values from 6 to 4.7 were able to compete with [125I]-labeled FSH for receptor binding in a parallel fashion. Thus, the observed shift in the PAG-IEF FSH profiles to more basic (and biologically active) forms may represent a change in the composition of AP FSH that serves an important role in the maturation process leading to ovulatory cyclicity.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Breed differences in clearance of porcine FSH in hypophysectomized rats

Extracts of anterior pituitary (AP) glands were infused i.v. into hypophysectomized male rats followed by sequential sampling of blood for 120 min. Determination of follicle-stimulating hormone (FSH) concentrations established that FSH from Chinese Meishan males decreased in the circulation of rats more slowly than FSH in extracts of AP from crossbred occidental pigs (P<0.003). Additionally, FSH from AP extracts of castrated males disappeared somewhat more slowly (P<0.06) than FSH from extracts of boars. Evaluation of FSH by bioassay and radioimmunoassay yielded similar concentrations in AP from Meishan and crossbred boars. Serum testosterone concentrations increased with time through 90 min after infusion of AP, but the rate of increase of testosterone was not related to amount of luteinizing hormone (LH) that was administered indicating LH receptor saturation. Unexpectedly, the rate of increase in testosterone was more rapid with AP extracts from boars than with extracts from castrated males. Observations from the current study imply structural alterations of FSH in the AP of Meishan males relative to crossbred males allowing sustained concentrations in the circulation, and this FSH possesses similar activation of the FSH receptor. The amount of LH in the AP extracts saturated the LH receptors of the hypophysectomized male rats, but some factor in extracts of boars differed from those of castrated males.     

Studies of the effectiveness of gonadotrophin-releasing hormone, steroids and follicular fluid in modulating ovine gonadotrophin output in vivo and in vitro

Gonadotrophin-releasing hormone (GnRH) readily stimulated LH output by sheep pituitary cells in vitro, and raised plasma LH concentrations in vivo in sheep, in a dose-dependent fashion. However, increases in FSH levels were only marginal by comparison. Dose-dependent decreases in sheep pituitary cell FSH output and in plasma FSH concentrations were caused by sheep follicular fluid and oestradiol-17 beta in vitro, and by bovine follicular fluid and oestradiol benzoate in vivo. In contrast, LH concentrations were only reduced slightly at the higher doses of these reagents. Cumulative suppressive effects of follicular fluid and oestradiol-17 beta (oestradiol benzoate) on FSH levels were observed both in vitro and in vivo. The transient positive feedback effect of oestradiol benzoate on FSH output negated the suppressive effect of bovine follicular fluid on plasma FSH concentrations. Progestagens, androgens and catechol oestrogens also suppressed mean FSH output in vitro, though not as effectively as oestradiol-17 beta. While only 1-5 pg/ml of oestradiol-17 beta was needed to suppress significantly mean FSH output in vitro, greater than 500 pg/ml of the other steroids was required. Seminal plasma inhibin-like peptide failed to suppress mean FSH output by cultured sheep pituitary cells at doses from 1 pg/ml to 500 ng/ml. At higher doses, both FSH and LH output was suppressed and this was accompanied by morphological deterioration of the cells. It is suggested that, to raise plasma FSH concentrations with a view to increasing ovulation rates in sheep, the development of means to reduce the negative feedback effects of steroids, notably oestradiol-17 beta, and inhibin on FSH secretion may be a more appropriate pharmacological strategy than increasing pituitary exposure to GnRH.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

A study on expression of FSH and its effects on the secretion of insulin and glucagon in rat pancreas

Studies indicate that many tissues could express follicle-stimulating hormone (FSH) besides pituitary. New functions of FSH are also been recognized beyond reproduction regulation. However, no report has been made about the expression and function of FSH in rat pancreas yet. Dual-labeled immunofluorescence stain, in situ hybridization and dual-labeled immunohistochemistry stain in adjacent sections were used to study the expression of FSH and its receptor, and co-localization of FSH with gonadotropin-releasing hormone (GnRH) receptor in rat pancreas. Tissue incubation and enzyme-linked immunosorbant assay (ELISA) were used to study the effects of FSH on the secretion of insulin and glucagon in rat pancreas in vitro. The results showed that rat pancreas could express FSH and its receptor, some of islet cells co-expressed FSH and its receptor, some of islet cells co-expressed FSH and GnRH receptor. FSH has the same bidirectional regulation effects on insulin and glucagon in vitro. These data suggested that rat pancreas is a target organ of FSH, and GnRH might regulate FSH through GnRH receptor in rat pancreas. FSH might regulate the endocrine function of rat pancreas through FSH receptor.     

Surges of FSH during the follicular and early luteal phases of the estrous cycle in heifers

Surges of FSH were characterized in each of 12 Holstein heifers using a computerized cycle detector program, and as mean changes averaged over all heifers. Blood samples were collected 6 times a day at 4-h intervals beginning at late diestrus. Concentrations of FSH were adjusted relative to the preovulatory LH peak (Hour 0) and profiled beginning 48 h before and ending 120 h after the LH peak. Peak concentrations of FSH and LH occurred synchronously in 11 of 12 (92%) heifers, and only a 4-h interval separated peak concentrations in the remaining heifer. The FSH surge that was synchronous with the LH surge was designated FSH Surge 1 and was used as a reference to designate other FSH surges. Surge -1 of FSH was detected in 58% of the heifers at mean Hour -21.2, and Surges 2, 3 and 4 were detected in 92%, 92% and 75% of the heifers, respectively, at mean Hours 25.1, 57.8 and 78.7. Mean peak levels and duration of FSH Surges-1, 2, 3 and 4 were significantly lower than for FSH Surge 1. Mean concentrations of FSH significantly increased and decreased before and after the LH peak, resulting from the synchrony between FSH Surge 1 and the LH surge in individual heifers. Additionally, there was a tendency (P < 0.08) for a second and third increase in mean FSH concentrations at Hours 24 and 60, which was attributed to FSH Surges 2 and 3 that occurred in individuals. Peak FSH concentrations of Surge 2 occurred (mean, Hour 25.1) within 8 h of maximal mean concentrations at Hour 24 in 91% of the heifers. Correspondingly, peak FSH concentrations of Surge 3 occurred (mean, Hour 57.8) within 8 h of maximal mean concentrations at Hour 60 in 64% of the heifers. Surges -1 and 4 of FSH occurred less frequently and at various times within and among heifers compared with Surges 1 to 3; therefore, they were not detected as mean increases in FSH concentrations but were masked as a result of concentrations being averaged over all heifers. In summary, FSH surges were detected in individual heifers before and after the combined FSH/LH surge. The interpeak intervals for FSH Surges 1 to 2 (25 h), 2 to 3 (33 h) and 3 to 4 (21 h) suggests a rhythmic nature to the surges.     

Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro

Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF1 mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (> or =20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.     

Insulin alters the effects of follicle stimulating hormone on aromatase in bovine granulosa cells in vitro

It is known that follicle-stimulating hormone (FSH) and insulin stimulate estradiol secretion from cultured non-luteinizing granulosa cells. The interaction between these hormones is less well understood. Granulosa cells from small (2-4 mm) bovine follicles were cultured in serum-free medium to determine if cytochrome P450 aromatase activity is regulated by FSH in the presence of different concentrations of insulin. Insulin significantly stimulated aromatase activity in the absence of FSH. There was a significant interaction between insulin and FSH on aromatase activity, such that FSH stimulated activity at low (0.5, 1 and 10 ng/ml) doses of insulin, whereas at higher (100 ng/ml) doses of insulin FSH failed to stimulate aromatase activity. To determine if the lack of a response to FSH with higher doses of insulin is related to gene expression, the effect of FSH on P450 aromatase mRNA levels was measured. An 'uncoupling' of mRNA and enzyme activity was observed for cells cultured with 100 ng/ml insulin, as FSH significantly increased P450 aromatase mRNA abundance without affecting estradiol secretion or aromatase activity. We conclude that in the presence of high doses of insulin, FSH decreases aromatase activity, and an uncoupling of P450 aromatase mRNA and aromatase activity occurs. This may have implications for infertility treatments when there is a risk of hyperinsulinemia.     

Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.     

Expression of biologically active human follitropin in Chinese hamster ovary cells

To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.     

Changes in serum FSH concentrations in the pig during development

Serum FSH concentrations were measured in fetal and prepubertal pigs between 40 days postcoitum and 25 weeks after birth. In addition, serum FSH was estimated in prepubertal, unilaterally cryptorchid, freemartin and castrated pigs. The average serum FSH concentrations in male and female fetuses was low (less than 2 ng/ml) until 80 days p.c. During the remaining fetal period, concentrations in females were elevated (7.9 +/- 0.4 ng/ml) and remained fairly constant after birth (16.3 +/- 0.8 ng/ml). In the male, serum FSH concentrations gradually rose to 22.5 +/- 5.5 ng/ml during the first 3 weeks after birth and declined thereafter. The changes in FSH concentrations in male pigs are reflected in gonadal-development. In contrast, in fetal and prepubertal females, ovarian development seems not to be influenced by changes in serum FSH concentrations. Unilateral cryptorchidism did not affect serum FSH concentrations. After castration, however, concentrations rose significantly. In freemartin pigs concentrations were similar to those in female pigs.