FSH Human

What is human FSH and what molecular characteristics distinguish it from other species?

Human FSH is a heterodimeric glycoprotein hormone consisting of noncovalently linked α and β subunits that plays a crucial role in the regulation of reproduction . It stimulates the growth of immature follicles in ovaries and primary spermatocytes in testes .

The human FSH receptor demonstrates species-specific ligand recognition that differs from rodent receptors. Research has shown that while human, rat, and ovine FSH can compete for rat testicular FSH receptor binding, only human FSH (hFSH) and rat FSH (rFSH) interact effectively with the recombinant human FSH receptor . This specificity must be considered when designing cross-species experiments or interpreting results from animal models.

Additionally, expression studies have revealed that human FSH receptor mRNA transcripts exist in multiple sizes (7.0, 4.2, and 2.5 kilobases) specifically in human follicular phase ovary tissue, but not in corpus luteum or placenta .

How do FSH tests evaluate reproductive function and what markers should researchers monitor?

FSH blood tests measure the level of this hormone in the bloodstream and are valuable for evaluating sexual maturation, fertility issues, and menstrual problems . In research contexts, FSH is rarely studied in isolation, as human sex hormones work closely with one another. A comprehensive experimental design should include testing FSH along with luteinizing hormone (LH), estradiol, and testosterone to provide a more complete picture of reproductive hormone status .

When studying FSH action in experimental models, researchers should monitor:

• Serum FSH and related hormone levels

• Receptor binding parameters (Kd values)

• Downstream cyclic AMP (cAMP) accumulation

• Expression of specific marker genes like PTGS2, which has been shown to be significantly downregulated in follicular fluid sediment cells of non-conception patients

What are the biochemical properties of human FSH receptors that researchers should account for in experimental design?

Human FSH receptors demonstrate high affinity binding with a Kd value of approximately 1.7 × 10⁻⁹ M . After transfection of human FSH receptor cDNA into cell lines, radioligand receptor analysis reveals these high-affinity binding sites on the plasma membrane .

When designing binding experiments, researchers should note that:

• Both recombinant and wild-type hFSH can displace [¹²⁵I]hFSH binding, with ED₅₀ values of 25 and 70 ng/ml, respectively

• Human LH, human chorionic gonadotropin (hCG), and human thyroid-stimulating hormone (hTSH) do not compete effectively with FSH binding

• Functional coupling of the expressed human receptor with adenyl cyclase can be demonstrated by measuring extracellular cAMP accumulation, with an ED₅₀ of approximately 10 ng/ml

After ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two FSH-binding sites of 76 and 112 kilodaltons can be detected in transfected cells .

What gain-of-function genetic models exist for studying FSH action and how can they be applied?

Several gain-of-function (GOF) genetic models have been developed to study FSH action in physiological contexts. These models provide powerful tools for understanding FSH biology and developing new therapeutic approaches.

The first GOF mouse model for FSH was generated more than 25 years ago using a 10 kb human FSHB transgene . Since then, researchers have used various promoters to achieve both gonadotrope-specific and ectopic expression of FSH ligand .

Key FSH GOF models include:

• Transgenic hFSHB Expression Model: This model exhibits pituitary-specific transgene expression with no ectopic expression in non-pituitary tissues. FSH dimer secretion and pituitary HFSHB mRNA expression were higher in males than females, demonstrating conservation of regulatory elements across mice and humans .

• Constitutively Active FSHR Models: Mouse models harboring mutant versions of Fshr (D580H and D580Y) have been developed to identify phenotypic consequences of activating mutations. These models showed that ovary-specific expression of mutant FSHR resulted in multiple ovarian abnormalities, including hemorrhagic cysts, accelerated loss of immature follicles, and increased granulosa cell proliferation .

• FSHR Overexpression Model (TgFSHRwt): A comparative study using transgenic mice overexpressing wild-type human FSH receptor demonstrated that physiological responses to constitutively active mutant FSH receptors were due to the mutation itself and not receptor overexpression .

• Combined Models: FSH GOF models combined with Fshb null mice provide an efficient genetic rescue platform that can be used as an in vivo functional assay for testing bioactivities of FSH and FSH analogs .

How can researchers differentiate between FSH receptor activation by mutation versus receptor overexpression?

Distinguishing between effects caused by mutant receptor activity versus those resulting from receptor overexpression requires careful comparative studies. Research has demonstrated that this differentiation can be achieved by comparing transgenic mice expressing a constitutively active mutant FSH receptor (FSHR+) with those overexpressing wild-type human FSH receptor (TgFSHRwt) .

Methodologically, researchers should:

• Confirm similar levels of receptor expression by measuring radioactive ligand binding (e.g., ¹²⁵I-FSH binding to testicular membranes)

• Compare physiological parameters across both models and controls:

  • Testis weights and hormone levels (e.g., testosterone)

  • cAMP activity in target cells (basal and stimulated)

  • Receptor ligand-specificity using various hormones (e.g., hCG, TSH)

  • Expression of steroidogenic enzyme-encoding mRNAs (e.g., Cyp11a1, Star)

  • Cellular maturation status

In one comparative study, TgFSHRwt mice showed no difference in testis weights or serum testosterone levels compared to controls, while FSHR+ mice exhibited larger average testis weights and elevated testosterone concentrations. Although TgFSHRwt Sertoli cells showed higher cAMP activity in vitro, they did not display the increased basal activity observed in FSHR+ cells .

What are the effects of FSH on human follicular fluid-derived stromal cells and their practical research applications?

Research on human follicular fluid (FF) stromal cells has revealed that FSH treatment influences these cells in several ways that may be relevant to fertility outcomes. When characterizing FF-derived stromal cells treated with FSH, researchers found that:

The expression of vimentin and cadherin in FF stromal cells may be somewhat promoted by FSH treatment in both conceived and not-conceived patients . Mass spectrometry analysis identified 97 proteins secreted by FF stromal cells, related to stress response, positive regulation of apoptotic cell clearance, and embryo implantation .

These findings have practical applications in research on fertility mechanisms and potential therapeutic interventions.

What techniques are most effective for studying FSH receptor expression and function?

Several methodological approaches have proven effective for studying FSH receptor expression and function:

• cDNA Library Screening: Using fragments of known FSH receptor cDNA (e.g., rat FSH receptor) to screen human testicular cDNA libraries to obtain full-length receptor cDNA .

• Transfection Studies: Transfecting human cell lines (e.g., human fetal kidney cell line 293) with FSH receptor cDNA to study receptor properties .

• Radioligand Receptor Analysis: This approach identifies high-affinity FSH-binding sites on the plasma membrane and allows for determination of binding constants such as Kd values .

• Competition Binding Assays: Using labeled FSH (e.g., [¹²⁵I]hFSH) and various unlabeled hormones to assess receptor specificity .

• cAMP Accumulation Assays: Measuring extracellular cAMP levels after hormone treatment to assess functional coupling with adenyl cyclase .

• Reporter Gene Assays: Co-transfecting cells with FSH receptor expression plasmid and a luciferase reporter gene driven by a cAMP-responsive promoter to measure downstream signaling .

• Northern Blot Analysis: Using cRNA probes derived from receptor cDNA to detect FSH receptor mRNA transcripts in tissues .

• Ligand/Receptor Cross-Linking: Followed by SDS-PAGE analysis to detect FSH-binding protein complexes .

How should researchers characterize follicular fluid-derived stromal cells in FSH studies?

Characterization of follicular fluid-derived stromal cells should include:

• Surface Marker Expression: FF stromal cells are typically positive for mesenchymal stromal cell markers (CD90+, CD44+, CD166+) and HLA-ABC+. Additional markers to assess include CD73, CD13, CD146, CD105, CD56, and BMSC. There should be little to no expression of SUSD2, CD338, CD140b, and hematopoietic cell markers CD34 and CD45 .

• Morphological Assessment: Evaluate cell morphology with and without FSH treatment, although research suggests FSH has minimal impact on morphology .

• Proliferation Analysis: Measure proliferation rates, as FSH has been shown to lower proliferation in these cells .

• Gene Expression Analysis: Use real-time PCR to analyze the expression of ovarian follicle development and FSH response-related genes, including ovulatory cascade genes that may differ between conceived and not-conceived patients' FF stromal cells .

• Protein Expression: Assess the expression of structural proteins like vimentin and cadherin, which may be promoted by FSH treatment .

• Secretome Analysis: Mass spectrometry can identify proteins secreted by FF stromal cells, which relate to stress response, apoptotic cell clearance, and embryo implantation .

What are the current best practices for production of recombinant human FSH for research purposes?

Production of high-quality recombinant human FSH (rh-FSH) for research applications has evolved from traditional stainless steel vessel systems with serum-containing media to more modern approaches:

• Expression System Selection: Many biopharmaceutical companies are switching from traditional production systems to single-use (SU) bioreactors due to numerous advantages .

• Single-Use Bioreactors: These provide manufacturing advantages including fewer validation requirements, lower contamination risks, elimination of cleaning and sterilization steps, and rapid turn-around between batches .

• Process Optimization: Pure recombinant human gonadotropin processes (e.g., Gonapure) provide consistently high hormone production in upstream fed-batch processes .

• Scale Considerations: Upstream rh-FSH processes can be designed with different scales of single-use bioreactor systems to accommodate various research needs .

When comparing to earlier methods, researchers should note that treatments with urinary FSH have been largely replaced by rh-FSH, which offers several advantages including absence of luteinizing hormone activity, higher specific activity, and lower risk of infection .

How should researchers design experiments to study FSH receptor mutations and their physiological consequences?

When designing experiments to study FSH receptor mutations, researchers should consider the following methodological approach:

• Mutation Selection: Identify specific mutations of interest, such as activating mutations like D580H and D580Y in the FSH receptor .

• Expression Strategy: Consider different expression approaches:

  • Cell-specific expression using tissue-specific promoters (e.g., AMH promoter for ovary-specific expression)

  • Knock-in mutations to the endogenous receptor locus

  • Transgenic expression systems

• Functional Characterization: Assess:

  • Basal receptor activity

  • Ligand binding capacity

  • Ligand dependence

  • Promiscuity toward other hormones (e.g., LH/CG stimulation)

• Phenotypic Analysis: Evaluate tissue-specific and systemic effects:

  • For ovarian studies: follicle development, cyst formation, granulosa cell proliferation, hormone biosynthesis, fertility

  • Secondary effects on other tissues: mammary gland, pituitary, adrenal glands

• Comparative Analysis: Compare mutant phenotypes with receptor overexpression controls to distinguish mutation-specific effects from general overexpression effects .

• Genetic Background Considerations: Consider testing mutations on different genetic backgrounds, including receptor knockout backgrounds for rescue experiments .

This approach has been successfully used to identify phenotypic consequences of FSH receptor mutations, providing insights that can guide the search for similar phenotypes in patients carrying analogous mutations .

What are the potential applications of FSH research in addressing clinical reproductive challenges?

FSH research has several potential applications in addressing clinical reproductive challenges:

• Improved Infertility Treatments: Understanding FSH action at the molecular and cellular levels can lead to more effective and personalized fertility treatments . Research has shown that treatments with recombinant human FSH provide several advantages over urinary FSH, including absence of luteinizing hormone activity, higher specific activity, and lower risk of infection .

• Diagnostic Markers: Identification of differential gene expression patterns, such as the downregulation of PTGS2 in follicular fluid sediment cells of patients who did not conceive, could lead to new diagnostic markers for fertility outcomes .

• Novel Therapeutic Approaches: Insights from gain-of-function genetic models and their physiological consequences can potentially be used to develop new approaches for treating clinical conditions involving FSH .

• Precision Medicine: Understanding the variability in FSH response based on genetic factors could enable more tailored approaches to reproductive medicine.

How can researchers leverage FSH genetic models to develop new therapeutic strategies?

Researchers can leverage FSH genetic models to develop new therapeutic strategies through several approaches:

• Genetic Rescue Platforms: Combining FSH gain-of-function models with Fshb null mice provides a powerful genetic rescue platform that can be used as an efficient in vivo functional assay for testing bioactivities of FSH and FSH analogs .

• Constitutively Active Receptor Models: These models can be used to understand the physiological consequences of receptor activation independent of ligand binding, which may inform the development of small molecule activators of FSH signaling .

• Cell-Specific Expression Models: Models with tissue-specific expression of FSH or its receptor can help identify the direct effects of FSH on target tissues versus indirect systemic effects, enabling more targeted therapeutic approaches .

• Comparative Analysis: Studying species-specific differences in FSH regulation and action can help identify conserved mechanisms that are most likely to translate to human applications .

• Protein Engineering: Understanding the structure-function relationships of FSH and its receptor can guide the engineering of FSH variants with enhanced or selective activities .