Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

Metabolic transitions define spermatogonial stem cell maturation.

STUDY QUESTION: Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? SUMMARY ANSWER: Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. WHAT IS KNOWN ALREADY: Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. STUDY DESIGN, SIZE, DURATION: To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. LARGE SCALE DATA: New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. LIMITATIONS, REASONS FOR CAUTION: Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.

STUDY QUESTION: Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized processing/freezing for centers that do not have those capabilities? SUMMARY ANSWER: Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice. WHAT IS KNOWN ALREADY: Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient's tissue was donated to research and 75% was stored for patient's future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg-6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size. WIDER IMPLICATIONS OF THE FINDINGS: Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested. STUDY FUNDING/COMPETING INTEREST(S): Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.

Effect of hormone modulations on donor-derived spermatogenesis or colonization after syngeneic and xenotransplantation in mice.

BACKGROUND: Cytotoxic cancer treatments, such as irradiation, can cause permanent sterility in male mammals owing to the loss of spermatogonial stem cells. In animal models, spermatogenesis could be restored from transplanted spermatogonial stem cells. Previously, we showed that transient suppression of FSH, LH, and testosterone in the recipient with a gonadotropin-releasing hormone antagonist (GnRH-ant), given immediately after irradiation, enhanced spermatogenesis from transplanted spermatogonial stem cells in mice and monkeys. OBJECTIVES: To explore improvements in the preparation of the recipient for efficient and reliable spermatogenic recovery from spermatogonial stem cell transplantation, so that it can be used effectively in clinical practice. MATERIALS AND METHODS: In mouse recipients, we evaluated the effects of hormone suppression given after germ cell depletion was complete, which is a more clinically relevant model, and also the importance of total androgen ablation and maintenance of FSH levels. Three regimens, GnRH-ant, GnRH-ant plus flutamide (androgen receptor antagonist), and GnRH-ant plus FSH, were administered prior to and around the time of transplantation of testis cells from immature mice or from prepubertal monkeys. RESULTS: Treatment with GnRH-ant resulted in a fourfold increase in spermatogenic recovery from GFP-marked transplanted mouse cells. Total androgen ablation with the addition of flutamide, started two weeks before transplantation, did not further enhance recovery. Surprisingly, FSH supplementation, started around the time of transplantation, actually reduced spermatogenic recovery from transplanted spermatogonial stem cells in GnRH-ant-treated mice. When prepubertal monkey testicular cells were transplanted into nude mice that were given the same hormone treatments, the numbers of donor-derived colonies were independent of hormone treatment. DISCUSSION AND CONCLUSION: The enhancements in spermatogenic recovery may only occur when syngeneic or closely related donor-recipient pairs are used. These results are useful in further investigations in choosing a hormone suppression regimen in combination with spermatogonial transplantation as a treatment to restore fertility in primates after cytotoxic therapy.

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

STUDY QUESTION: Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? SUMMARY ANSWER: In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. WHAT IS KNOWN ALREADY: In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. STUDY DESIGN, SIZE, AND DURATION: We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. PARTICIPANTS/MATERIALS SETTING METHODS: Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. MAIN RESULTS AND THE ROLE OF CHANCE: Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. WIDER IMPLICATIONS OF THE FINDINGS: We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys.

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin-releasing hormone antagonist (GnRH-ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared with radiation-only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH-ant-treated monkeys and (iv) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

Transgenic mice produced by retroviral transduction of male germ-line stem cells.

Male germ-line stem cells are the only cell type in postnatal mammals that have the capability to self-renew and to contribute genes to the next generation. Genetic modification of these cells would provide an opportunity to study the biology of their complex self-renewal and differentiation processes, as well as enable the generation of transgenic animals in a wide range of species. Although retroviral vectors have been used as an efficient method to introduce genes into a variety of cell types, postnatal male germ-line stem cells have seemed refractory to direct infection by these viruses. In addition, expression of genes transduced into several types of stem cells, such as embryonic or hematopoietic, is often attenuated or silenced. We demonstrate here that in vitro retroviral-mediated gene delivery into spermatogonial stem cells of both adult and immature mice results in stable integration and expression of a transgene in 2-20% of stem cells. After transplantation of the transduced stem cells into the testes of infertile recipient mice, approximately 4.5% of progeny from these males are transgenic, and the transgene is transmitted to and expressed in subsequent generations. Therefore, there is no intrinsic barrier to retroviral transduction in this stem cell, and transgene expression is not extinguished after transmission to progeny.

Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility.

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2-3 months after transplantation into adult tubules, was similar ( approximately 0.5 mm(2)). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.

Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells.

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules alpha6-integrin, alphav-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for alpha6-integrin, and negative or low alphav-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

Transcriptional activation of the decidual/trophoblast prolactin-related protein gene.

The decidual/trophoblast PRL-related protein (d/tPRP) is dually expressed by decidual and trophoblast cells during pregnancy. We have characterized the proximal d/tPRP promoter responsible for directing d/tPRP expression in decidual and trophoblast cells. We have demonstrated that the proximal 93 bp of d/tPRP 5'-flanking DNA are sufficient to direct luciferase gene expression in primary decidual and Rcho-1 trophoblast cells, but not in fibroblast, undifferentiated uterine stromal cells or trophoblast cells of a labyrinthine lineage. The 93-bp d/tPRP promoter was also sufficient to direct differentiation-dependent expression in trophoblast giant cells. Mutational analysis demonstrated the differential importance of activating protein-1 and Ets regulatory elements (located within the proximal 93 bp of d/tPRP 5'-flanking DNA) for activation of the d/tPRP promoter in decidual vs. trophoblast cells. Disruption of the activating protein-1 regulatory element inhibited d/tPRP promoter activity by more than 95% in decidual cells, and approximately 80% trophoblast cells. Disruption of the Ets regulatory element reduced d/tPRP promoter activity by approximately 50% in decidual cells, while inactivating the d/tPRP promoter in trophoblast cells. Protein interactions with the trophoblast Ets regulatory element were shown to be cell type specific and to change during trophoblast giant cell formation. In conclusion, a 93-bp region of the d/tPRP promoter is shown to contain regulatory elements sufficient for gene activation in decidual and trophoblast cells.

A new member of the mouse prolactin (PRL)-like protein-C subfamily, PRL-like protein-C alpha: structure and expression.

In this study, we establish the presence of a unique member of the PRL-like protein-C (PLP-C) subfamily in the mouse, PLP-C alpha, characterize its complementary DNA and gene, and map its chromosomal location and pattern of expression during pregnancy. Mouse PLP-C alpha encodes for a 239 amino acid protein and possesses from 69-71% identity with rat PLP-C, PLP-Cv, PLP-D, and PLP-H. Another feature characteristic of PLP-C subfamily members that is also present in mouse PLP-C alpha is a 6-exon/5-intron gene structure including an aromatic domain encoded by exon 3. Southern analysis with mouse and rat PLP-C subfamily probes suggested the existence of a single mouse PLP-C alpha gene. Mouse PLP-C alpha maps to chromosome 13 along with other members of the mouse PRL family. Expression of mouse PLP-C alpha increases dramatically as gestation advances and is restricted to spongiotrophoblast and trophoblast giant cells of the junctional zone. In summary, we have established the presence of a new PLP-C subfamily member in the mouse and demonstrated its similarity in structure and expression to rat PLP-C subfamily members. This level of conservation between species expands the biological significance of the PLP-C subfamily and provides additional opportunities for genetically evaluating its function.

Identification of two new members of the mouse prolactin gene family.

The prolactin (PRL) family consists of a collection of genes expressed in the uterus, placenta, and anterior pituitary. These cytokines/hormones participate in the control of maternal-fetal adaptations to pregnancy. In this report, we establish the presence of two new members of the mouse PRL family. Novel expressed sequence tags (ESTs) with significant homology to PRL were isolated from embryonic, ectoplacental cone, and placental cDNA libraries. The cDNAs were sequenced and compared to other members of the PRL family. The two new cDNAs were assigned to the PRL family based on sequence homology and were referred to as PRL-like protein-F (PLP-F) and PRL-like protein-G (PLP-G). PLP-F cDNA encodes for a predicted 267 amino acid protein containing a 30 amino acid signal peptide and three putative N-linked glycosylation sites. PLP-G cDNA encodes for a predicted 266 amino acid protein containing a 30 amino acid signal peptide and six putative N-linked glycosylation sites. Sequence alignments of these proteins with other members of the PRL family suggest some unique features. Both sequences contain an extra amino acid segment located between exons two and three of the prototypical PRL gene and a nine amino acid carboxy terminal extension. PLP-F contained an additional 15 amino acid region situated between exons four and five of the prototypical PRL gene. Both PLP-F and PLP-G mRNAs were expressed in the placenta but not in other tissues (uterus, brain, thymus, heart, lung, diaphragm, liver, kidney, and ovary). In summary, the two newly identified members share approximately 50% amino acid sequence identity, are specifically expressed in the placenta, and represent a new subfamily within the PRL family.

Homologues for prolactin-like proteins A and B are present in the mouse.

The prolactin (PRL) family consists of a collection of proteins expressed in the uterus, placenta, and anterior pituitary. These cytokines/hormones are hypothesized to control maternal-fetal adaptations to pregnancy. Establishment of mouse models for members of the PRL family expands the experimental repertoire available for investigations on their biological activities. In this report, we establish the presence of mouse homologues for two rat members, PRL-like protein-A (PLP-A) and PLP-B. We present data on their cDNAs and describe aspects of their expression in uteroplacental tissues. A mouse genomic DNA fragment was found to hybridize with a rat PLP-A cDNA. Perusal of the National Center for Biotechnology Information dbEST database resulted in the identification of several putative mouse PLP-A cDNAs and a single putative mouse PLP-B cDNA. The cDNAs were obtained from the IMAGE consortium and Research Genetics and sequenced, and their corresponding mRNAs and proteins were characterized. Overall, mouse PLP-A and PLP-B showed considerable similarities with rat PLP-A and PLP-B in both structure and expression. PLP-A was expressed in both trophoblast giant cells and spongiotrophoblast cells, whereas PLP-B was expressed in decidual and spongiotrophoblast cells. However, some notable exceptions were evident. Mouse PLP-A contained a single putative N-linked glycosylation site and consisted of a single 29-kDa protein species, whereas rat PLP-A contained two putative N-linked glycosylation sites and consisted of two protein species, of 29 and 33 kDa. Subtle differences in the expression patterns in the mouse and rat are also apparent. In summary, we have established the presence of PLP-A and PLP-B in the mouse. The findings expand our knowledge of these two cytokines/hormones and provide additional strategies for studying their function.

Identification and characterization of a mouse homolog for decidual/trophoblast prolactin-related protein.

Decidual/trophoblast PRL-related protein (d/tPRP) is one member of a large placental PRL gene family composed of at least nine members in the rat and four in the mouse. Only placental lactogen I and II have been characterized in both rat and mouse. The identification of mouse homologs for rat placental PRL family members will facilitate gene manipulation studies aimed at identifying functions for these hormones. In this report, we establish the presence of d/tPRP in the mouse and characterize its complementary DNA, protein, and pattern of expression during mouse gestation. Evaluation of the National Center for Biotechnology Information database of expressed sequence tags resulted in the identification of several mouse complementary DNA clones exhibiting significant homology to rat d/tPRP. One of these clones was obtained from IMAGE Consortium and Research Genetics for further investigation. The full-length mouse clone was found to have an 81% nucleotide homology with rat d/tPRP and to encode a 239-amino acid protein. Like rat d/tPRP, the mouse protein contains two putative N-linked glycosylation sites and six homologously located cysteine residues. Mouse d/tPRP maps to chromosome 13 along with other members of the mouse PRL family. Like the rat, mouse d/tPRP messenger RNA and protein are expressed by antimesometrial decidual cells and spongiotrophoblast and trophoblast giant cells in the junctional zone of the placenta. In summary, we have established the presence of d/tPRP in the mouse and demonstrated its similarity in structure and pattern of expression to rat d/tPRP. This level of conservation between species expands the biological significance of d/tPRP during pregnancy and provides additional opportunities for evaluating its function.

Decidual/trophoblast prolactin-related protein: characterization of gene structure and cell-specific expression.

Decidual/trophoblast PRL-related protein (d/tPRP) is a member of the PRL gene family and is dually expressed in uterine and placental tissues in a highly coordinated pattern during pregnancy. In the present study, we describe the isolation and characterization of the d/tPRP gene. A lambda DASH II Wistar-Kyoto rat genomic library was screened with a labeled d/tPRP complementary DNA, resulting in the isolation of two phage clones, RGLd-41 [17.7 kilobases (kb)] and RGLd-42 (15.8 kb). RGLd-41 alone was found to contain the full-length d/tPRP gene and was used for subsequent analyses. The d/tPRP gene possesses a six-exon, five-intron organization. Relative to other highly conserved members of the PRL gene family, d/tPRP contains a single small additional exon (exon 3) situated between exons 2 and 3 of the prototypical PRL gene. The region corresponding to exon 3 of d/tPRP encodes for a unique amino acid region found in a subset of PRL family members. A reverse transcription-PCR (RT-PCR) tissue survey for d/tPRP messenger RNA revealed that d/tPRP expression was restricted to decidual and trophoblast tissues. A single transcription start site 65 bp upstream of the initiation codon was identified in decidual tissue, whereas multiple transcription start sites ranging from 61-66 bp upstream of the initiation codon were detected in placental tissue. Various tissue culture systems (primary cultures and cell lines) were evaluated for d/tPRP expression and activation of a 3.96-kb d/tPRP promoter-luciferase reporter construct. Decidual, spongiotrophoblast, and trophoblast giant cell populations expressed d/tPRP and were capable of activating the d/tPRP promoter-reporter construct, whereas other cell types were ineffective. Limited d/tPRP promoter activation was noted in uterine stromal cell lines. In summary, d/tPRP possesses a unique six-exon, five-intron gene structure and exhibits cell-specific expression that is regulated at least in part by a 3.96-kb 5'-flanking region.

Dual expression of prolactin-related protein in decidua and trophoblast tissues during pregnancy in rats.

Decidual prolactin-related protein (dPRP) is a member of the prolactin gene family and is abundantly expressed in the rat deciduum. Previously, dPRP was shown to associate with heparin-containing molecules and was found to reside, at least in part, within the decidual extracellular matrix, where it was postulated to influence decidual cells and other cell types. The purpose of this investigation was to identify the cellular origin and the temporal and regional characteristics of dPRP expression in the rat uterus during pregnancy. Protein expression was evaluated by Western blot analysis, immunoprecipitation, and immunocytochemistry; dPRP mRNA expression was assessed by reverse transcriptase polymerase chain reaction and in situ hybridization. Decidual PRP was first detected at Day 6 of pregnancy or pseudopregnancy. Expression increased with the growth of the deciduum and then declined coincident with regression of decidual tissue. Throughout the first half of pregnancy or pseudo-pregnancy, dPRP and mRNA were predominantly localized to the antimesometrial deciduum of the developing conceptus. During the second half of gestation, expression also appeared in the chorioallantoic placenta. Trophoblast giant cells and spongiotrophoblast cells within the junctional zone of the chorioallantoic placenta expressed dPRP, as did the Rcho-1 trophoblast cell line. In conclusion, dPRP production is elevated from implantation until parturition through the participation of decidual (early pregnancy) and trophoblastic (late pregnancy) tissues.