Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: an approach to non-surgical sterilization

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades, but this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability we discuss how these reagents might be adjusted in order to increase target cell ablation and improve their efficacy as sterilization agents.

3 Introduction 10mg/3 mL and placed in a reaction vessel. The maleimide-Auristatin solution was then spectrometry, as above, before the product was freeze dried under vacuum and stored as a 292 powder at -20°C. 293 In vivo pilot study FSH2Menadione with LH2Auristatin 294 Four treatment groups of ten 55-day-old male Swiss male CD1 mice were injected 295 intraperitoneally. Treatments included (1): (i) 300 µL vehicle (30% Solutol (Kolliphor)/PBS), 296 (2)ii) 300 µL/30 g 14.5 mM FSH2Menadione, (3) iii)100 µL/30g 425 µM LH2Aur, or (4)and (iv) approximately 13 days of pregnancy, females were euthanized weighed, embryos and 307 resorption moles were counted, ovaries were collected, weighed, and corpora lutea counted. 308 After the mating period, at ten weeks post-injection, all males were euthanized, weighed and 309 blood collected by cardiac puncture. Blood samples from each animal were decanted into 310 EDTA-coated tubes and plasma was separated (10 min, 400 g Immediately after dissection, epididymal spermatozoa were also collected for analysis 326 of motility, vitality, DNA strand breaks (HALO), DNA fragmentation (SCSA), the presence 327 of reactive oxygen species (MSR and DHE) and oxidative DNA adducts (anti-8-OH-dG). For 328 these assays, three incisions were made in the cauda epididymides with a surgical blade before 329 placing the tissue in prewarmed Biggers-Whitten-Whittingham (BWW) medium (31) with 5 330 U/ml penicillin, and 5 mg/mL streptomycin, pH 7.4, at 37°C. Spermatozoa were allowed to 331 swim-out for 10 min before being collected into clean tubes. Sperm motility was assessed 332 using a HTM-IVOS II Computer Assisted Sperm Analysis and sperm vitality was assessed using 333 the eosin exclusion test (32) and viewed using a light microscope at 40× magnification. At

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Design of LH receptor binding peptides 380 A LH receptor binding peptide (hereinafter referred to as LHa) was designed based 381 on equivalent residues to those incorporated into a previously developed FSH peptide mimic 382 (24). Specifically, the LHa peptide incorporated amino acid residues mapping to LH β-subunit 383 96-107 (Table 1). An additional three LH peptides were also designed based on sequences 384 that confer specific receptor binding. These were designated L57, L95 and L101 depending on   L101peptide, means that a non-ideal mixed product of mono-conjugated and bi-conjugated  (Table 1), in vitro selectivity (Fig 2B and 2C) and similar in vivo performance 521 to other assessed peptides (Fig 3B), we selected the LHa peptide as the preferred candidate 522 for delivery of Auristatin to the testes.  FSH2Menadione administration alone was not sufficient to compromise male fertility.

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In fact, all five males in the FSH2Menadione treatment group were fertile, with each of the 690 ten females housed with these males becoming pregnant and carrying live embryos at 13 days 691 post-mating (Fig 7A). Two of the females that had been mated with males from this treatment 692 group exhibited one resorption mole each, with none detected in the remaining eight dams.

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In contrast to the above, all five males in the vehicle treatment group proved to be fertile with 694 those females that became pregnant having at least 11 (up to 17) and an average of 13.5 695 embryos present at 13 days, none had resorption moles (Fig 7A).   Fig 1), displayed higher binding capability than either L101-FITC or LHa-FITC in vitro. 730 Since both the "seatbelt" and the βL2 loop regions of LHβ have been implicated in ligand 731 binding to the cognate LH receptor(23, 42), it is considered likely that differences in in vitro 732 peptide binding efficacy are attributed to their unique physicochemical properties. L95 concentrations (Fig 2B). This increase in binding capability and lack of specificity at higher

Supporting Information
and then Auristatin is released from PABC by 1,6-elimination. Structures drawn using