Role of Follicle-Stimulating Hormone in Spermatogenesis

Spermatogenesis is a concerted sequence of events during maturation of spermatogonia into spermatozoa. The process involves differential gene-expression and cell-cell interplay regulated by the key endocrine stimuli, i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-stimulated testosterone. FSH affects independently and in concert with testosterone, the proliferation, maturation and function of the supporting Sertoli cells that produce regulatory signals and nutrients for the maintenance of developing germ cells. Rodents are able to complete spermatogenesis without FSH stimulus, but its deficiency significantly decreases sperm quantity. Men carrying loss-of-function mutation in the gene encoding the ligand (FSHB) or its receptor (FSHR) present, respectively, with azoospermia or suppressed spermatogenesis. Recently, the importance of high intratesticular testosterone concentration for spermatogenesis has been questioned. It was established that it can be completed at minimal intratesticular concentration of the hormone. Furthermore, we recently demonstrated that very robust constitutive FSHR action can rescue spermatogenesis and fertility of mice even when the testosterone stimulus is completely blocked. The clinical relevance of these findings concerns a new strategy of high-dose FSH in treatment of spermatogenic failure

Extragonadal LH/hCG action-Not yet time to rewrite textbooks

Gonadotropins are indispensable in both sexes in the regulation of gonadal sex steroid production and gametogenesis. In addition to their well-established classical actions, numerous recent publications have indicated the presence and function of luteinizing hormone/chorionic gonadotropin receptors (LH/hCG-R) in a variety of extragonadal tissues. However, the physiological significance of such effects has remained unclear. We have generated two genetically modified mouse models, one with excessive production of hCG and the other with targeted disruption of LH/hCG-R gene, and used them to address the functions of LH and hCG. Numerous gonadal and extragonadal phenotypes were found in the models with the two extremes of LH/hCG action. However, when the extragonadal effects were scrutinized in greater detail, they all appeared to arise through modification of gonadal function, either through enhanced or inhibited response to LH/hCG stimulation. Hence, further evidence is needed before the extragonadal LH/hCG-R expression can be considered functionally significant.Fil: Pakarainen, Tomi. University of Turku; FinlandiaFil: Ahtiainen, Petteri. University of Turku; FinlandiaFil: Zhang, Fu Ping. University of Helsinki; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Huhtaniemi, Ilpo. University of Turku; Finlandia. IMperial College London; Reino Unid

Extragonadal FSHR Expression and Function—Is It Real?

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable “in house” FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls

Role of Follicle-Stimulating Hormone in Spermatogenesis

Spermatogenesis is a concerted sequence of events duringmaturation of spermatogonia into spermatozoa. The process involves differential gene-expression and cell-cell interplay regulated by the key endocrine stimuli, i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-stimulated testosterone. FSH affects independently and in concert with testosterone, the proliferation, maturation and function of the supporting Sertoli cells that produce regulatory signals and nutrients for the maintenance of developing germ cells. Rodents are able to complete spermatogenesis without FSH stimulus, but its deficiency significantly decreases sperm quantity. Men carrying loss-of-function mutation in the gene encoding the ligand (FSHB) or its receptor (FSHR) present, respectively, with azoospermia or suppressed spermatogenesis. Recently, the importance of high intratesticular testosterone concentration for spermatogenesis has been questioned. It was established that it can be completed at minimal intratesticular concentration of the hormone. Furthermore, we recently demonstrated that very robust constitutive FSHR action can rescue spermatogenesis and fertility of mice even when the testosterone stimulus is completely blocked. The clinical relevance of these findings concerns a new strategy of high-dose FSH in treatment of spermatogenic failure

The Roles of Luteinizing Hormone, Follicle-Stimulating Hormone and Testosterone in Spermatogenesis and Folliculogenesis Revisited

Spermatogenesis and folliculogenesis involve cell-cell interactions and gene expression orchestrated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH regulates the proliferation and maturation of germ cells independently and in combination with LH. In humans, the requirement for high intratesticular testosterone (T) concentration in spermatogenesis remains both a dogma and an enigma, as it greatly exceeds the requirement for androgen receptor (AR) activation. Several data have challenged this dogma. Here we report our findings on a man with mutant LH beta subunit (LH beta) that markedly reduced T production to 1-2% of normal., but despite this minimal LH stimulation, T production by scarce mature Leydig cells was sufficient to initiate and maintain complete spermatogenesis. Also, in the LH receptor (LHR) knockout (LuRKO) mice, low-dose T supplementation was able to maintain spermatogenesis. In addition, in antiandrogen-treated LuRKO mice, devoid of T action, the transgenic expression of a constitutively activating follicle stimulating hormone receptor (FSHR) mutant was able to rescue spermatogenesis and fertility. Based on rodent models, it is believed that gonadotropin-dependent follicular growth begins at the antral stage, but models of FSHR inactivation in women contradict this claim. The complete loss of FSHR function results in the complete early blockage of folliculogenesis at the primary stage, with a high density of follicles of the prepubertal type. These results should prompt the reassessment of the role of gonadotropins in spermatogenesis, folliculogenesis and therapeutic applications in human hypogonadism and infertility.</p

REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.</p

Mouse models of altered gonadotrophin action: insight into male reproductive disorders

The advent of technologies to genetically manipulate the mouse genome has revolutionised research approaches, providing a unique platform to study the causality of reproductive disorders in vivo. With the relative ease of generating genetically modified (GM) mouse models, the last two decades have yielded multiple loss-of-function and gain-of-function mutation mouse models to explore the role of gonadotrophins and their receptors in reproductive pathologies. This work has provided key insights into the molecular mechanisms underlying reproductive disorders with altered gonadotrophin action, revealing the fundamental roles of these pituitary hormones and their receptors in the hypothalamic–pituitary–gonadal axis. This review will describe GM mouse models of gonadotrophins and their receptors with enhanced or diminished actions, specifically focusing on the male. We will discuss the mechanistic insights gained from these models into male reproductive disorders, and the relationship and understanding provided into male human reproductive disorders originating from altered gonadotrophin action.Fil: Jonas, Kim C.. Imperial College London; Reino UnidoFil: Oduwole, Olayiwola O.. Imperial College London; Reino UnidoFil: Peltoketo, Hellevi . University of Oulu; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Huhtaniemi, Ilpo T. . Imperial College London; Reino Unido. University of Turku; Finlandi

Hyperprolactinemia induced by hCG leads to metabolic disturbances in female mice

The metabolic syndrome is a growing epidemic; it increases the risk for diabetes, cardiovascular disease, fatty liver, and several cancers. Several reports have indicated a link between hormonal imbalances and insulin resistance or obesity. Transgenic (TG) female mice overexpressing the human chorionic gonadotropin β-subunit (hCGβ+ mice) exhibit constitutively elevated levels of hCG, increased production of testosterone, progesterone and prolactin, and obesity. The objective of this study was to investigate the influence of hCG hypersecretion on possible alterations in the glucose and lipid metabolism of adult TG females. We evaluated fasting serum insulin, glucose, and triglyceride levels in adult hCGβ+ females and conducted intraperitoneal glucose and insulin tolerance tests at different ages. TG female mice showed hyperinsulinemia, hypertriglyceridemia, and dyslipidemia, as well as glucose intolerance and insulin resistance at 6 months of age. A 1-week treatment with the dopamine agonist cabergoline applied on 5-week-old hCGβ+ mice, which corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, effectively prevented the metabolic alterations. These data indicate a key role of the hyperprolactinemia-induced gonadal dysfunction in the metabolic disturbances of hCGβ+ female mice. The findings prompt further studies on the involvement of gonadotropins and prolactin on metabolic disorders and might pave the way for the development of new therapeutic strategies.Fil: Ratner, Laura Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Stevens, Guillermina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bonaventura, Maria Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lux, Victoria Adela R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Calandra, Ricardo Saul. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Huhtaniemi, Ilpo T.. University of Turku; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin